Genomic Organization and Developmental Fate of Adjacent Repeated Sequences in a Foldback DNA Clone of Tetrahymena thermophila

DNA sequence elimination and rearrangement occurs during the development of somatic cell lineages of eukaryotes and was first discovered over a century ago. However, the significance and mechanism of chromatin elimination are not understood. DNA elimination also occurs during the development of the somatic macronucleus from the germinal micronucleus in unicellular ciliated protozoa such as Tetrahymena thermophila. In this study foldback DNA from the micronucleus was used as a probe to isolate ten clones. All of those tested (4/4) contained sequences that were repetitive in the micronucleus and rearranged in the macronucleus. The presence of inverted repeated sequences was clearly demonstrated in one of them by electron microscopy. DNA sequence analysis showed that the left portion of this clone contains three tandem, directly repeated copies of a 340-bp sequence, a 120-bp portion of which appears in inverted orientation at a 1.6-kb distance. This clone, pTtFB1, was subjected to a detailed analysis of its developmental fate. Subregions were subcloned and used as probes against Southern blots of micronuclear and macronuclear DNA. We found that all subregions defined repeated sequence families in the micronuclear genome. A minimum of four different families was defined, two of which are retained in the macronucleus and two of which are completely eliminated. The inverted repeat family is retained with little rearrangement. Two of the families, defined by subregions that do not contain parts of the inverted repeat, one in the "loop" and one in the "right flanking region," are totally eliminated during macronuclear development--and contain open reading frames. A fourth family occurs in the "loop" region and is rearranged extensively during development. The two gene families that are eliminated are stable in the micronuclear genome but are not clustered together as evidenced by experiments in which DNAs from nullisomic strains are used to map family members to specific micronuclear chromosomes. The inverted repeat family is also stable in the micronuclear genome and is dispersed among several chromosomes. The significance of retained inverted repeats to the process of elimination is discussed.     

Organization of the micronuclear genome of oxytricha nova

In the hypotrichous ciliated protozoan Oxytricha nova, approximately 95% of the micronuclear genome, including all of the repetitive DNA and most of the unique sequence DNA, is eliminated during the formation of the macronuclear genome. We have examined the interspersion patterns of repetitive and unique and eliminated and retained sequences in the micronuclear genome by characterizing randomly selected clones of micronuclear DNA. Three major classes of clones have been defined: (1) those containing primarily unique, retained sequences; (2) those containing only unique, eliminated sequences; and (3) those containing only repetitive, eliminated sequences. Clones of type one and three document two aspects of organization observed previously: clustering of macronuclear destined sequences and the presence of a prevalent repetitive element. Clones of the second type demonstrate for the first time that eliminated unique sequence DNA occurs in long stretches uninterrupted by repetitive sequences. To further examine repetitive sequence interspersion, we characterized the repetitive sequence family that is present in 50% of the clones (class three above). A consensus map of this element was obtained by mapping approximately 80 phage clones and by hybridization to digests of micronuclear DNA. The repeat element is extremely large (approximately 24 kb) and is interspersed with both macronuclear destined sequences and eliminated unique sequences.     

Elimination of germ-line tandemly repeated sequences from the somatic genome of the ciliate Oxytricha fallax

The ciliated protozoa exhibit nuclear dimorphism. The genome of the somatic macronucleus arises from the germ-line genome of the micronucleus following conjugation. We have studied the fates of highly repetitious sequences in this process. Two cloned, tandemly repeated sequences from the micronucleus of Oxytricha fallax were used as probes in hybridizations to micronuclear and macronuclear DNA. The results of these experiments show: (1) the cloned repeats are members of two apparently unrelated repetitious sequence families, which each appear to comprise a few percent of the micronuclear genome, and (2) the amount of either family in the macronuclei from which our DNA was prepared is about 1/15 that found in an equal number of diploid micronuclei. Most, if not all, of the apparent macronuclear copies of these repeats can be accounted for by micronuclear contamination, which strongly suggests that these sequences are eliminated from the macronuclei and have no vegetiative function.     

Reassociation and dissociation of cytoplasmic membrane-associated DNA

The relationship between nuclear DNA and cytoplasmic membrane-associated DNA, extracted from a human lymphocyte cell line, was examined by DNA-DNA reannealing and by dissociation of renatured molecules. Up to 2% of the total cellular DNA is found in the cytoplasm as cytoplasmic membrane-associated DNA and of this 2%, approximately 70% is comprised of repeated sequences. These sequences are homologous to only about 4% of the repeated sequences of nuclear DNA. The repeat fraction of cytoplasmic membrane-associated DNA consists of sequences which are only moderately repeated. The number of copies in the average “family” could range from about 1500 copies to as few as 25 copies. A small rapidly reannealing portion of cytoplasmic membrane-associated DNA (C₀t < 4 × 10^?3) appears to consist of sequences derived from a single “family”.About 30% of cytoplasmic membrane-associated DNA reassociates slowly with a $\text{C}{}_0\text{t}\text{1}\text{2}$ value of 223 (unique cytoplasmic membrane-associated DNA). This fraction has homology with about 11% of the unique sequences of nuclear DNA. However, unique cytoplasmic membrane-associated DNA comprises only about 0·6% of the total cellular DNA. If it is assumed that each cell has the same amount of cytoplasmic membrane-associated DNA, homology with 11% of the unique sequences of nuclear DNA suggests that different cells may have different unique nucleotide sequences in the cytoplasm.     

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

Tlr elements are a novel family of ~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 ~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.     

Parental expression of the chromodomain protein Pdd1p is required for completion of programmed DNA elimination and nuclear differentiation

Thousands of DNA elimination events occur during somatic differentiation of many ciliated protozoa. In Tetrahymena, the eliminated DNA aggregates into submacronuclear structures containing the protein Pdd1p, a member of the chromodomain family. We disrupted somatic copies of PDD1, eliminating parental expression of the gene early in the sexual phase of the life cycle. Even though zygotic expression, from the undisrupted germline PDD1 copy, is activated before DNA elimination normally occurs, the somatic knockout cells suffer defects in DNA elimination, genome endoduplication, and nuclear resorption, and eventually die, demonstrating that PDD1 is essential and suggesting Pdd1p is directly involved in establishing a chromatin structure required for DNA elimination.     

Tandemly Repeated Sequence Families in Micronuclear DNA of the Ciliate Stylonychia pustulata1

One third of a collection of cloned Stylonychia pustulata micronuclear DNA PstI fragments were found to be of a similar size, consistent with their being members of a repetitious sequence family with a repeat size of about 160 base pairs. Cross-hybridization experiments confirmed that these small cloned fragments are related by sequence homology. Hybridization of the cloned repetitious sequences to PstI digested micronuclear DNA revealed a “ladder” of bands (step size = 160 base pairs), indicating that the repeats are found in tandem arrays. This is the first demonstration of highly repetitious, tandemly repeated sequences in a ciliated protozoan.     

Molecular characterization of Ascaris suum DNA and of chromatin diminution

A technique for the extraction of pure somatic (post-diminution) and germ line (pre-diminution) DNA from the parasitic nematode Ascaris is described. Uncontaminated post- and pre-diminution DNAs were sheared and reassociated to different C₀t values. Computer analysis of the complete reassociation kinetics determined that 33% of the germ line genome is eliminated during the process of chromatin diminution. The eliminated DNA is comprised of repetitive and unique sequences in an approx. 1:1 ratio.     

A germline restricted, highly repetitive DNA sequence in Paramyxineatami: an interspecifically conserved, but somatically eliminated, element

In some species of hagfish, the phenomenon of chromosome elimination occurs during embryogenesis. However, only two repetitive DNA families are known to be represented in chromosomes that are eliminated from somatic cells of the Japanese hagfish Eptatretus okinoseanus. Using molecular analyses, another germ line-restricted, highly repetitive DNA family has been detected in another Japanese hagfish, Paramyxine atami. The repeat unit of this family, which is 83?bp long, has been designated “EEPa1”, for Eliminated Element of P. atami 1. DNA filter hybridization using EEPa1 as a probe revealed that this family is shared among several species and is conserved in the germline DNA. Although eliminated, repetitive DNA that is shared interspecifically has not been reported in hagfish species, cases of chromatin diminution and chromosome elimination processes have been described previously in other organisms.The patterns and intensities of hybridization signals suggest that members of the repetitive DNA family defined by EEPa1 have undergone concerted molecular evolution.     

Rapid and repeatable elimination of a parental genome-specific DNA repeat (pGc1R-1a) in newly synthesized wheat allopolyploids

Recent work in the Triticum-Aegilops complex demonstrates that allopolyploidization is associated with an array of changes in low-copy coding and noncoding sequences. Nevertheless, the behavior and fate of repetitive DNA elements that constitute the bulk of nuclear DNA of these plant species is less clear following allopolyploidy. To gain further insight into the genomic events that accompany allopolyploid formation, we investigated fluorescence in situ hybridization (FISH) patterns of a parental-specific, tandem DNA repeat (pGc1R-1) on three sets of newly synthesized amphiploids with different parental species. It was found that drastic physical elimination of pGc1R-1 copies occurred in all three amphiploids in early generations. DNA gel-blot analysis confirmed the FISH data and estimates indicated that approximately 70-90% of the copies of the pGc1R-1 repeat family were eliminated from the amphiploids by the second to third selfed generations. Thus, allopolyploidy in Triticum-Aegilops can be accompanied by rapid and extensive elimination of parental-specific repetitive DNA sequences, which presumably play a role in the initial stabilization of the nascent amphiploid plants.     

Micronuclear DNA of Oxytricha nova contains sequences with autonomously replicating activity in Saccharomyces cerevisiae.

Oxytricha nova is a hypotrichous ciliate with micronuclei and macronuclei. Micronuclei, which contain large, chromosomal-sized DNA, are genetically inert but undergo meiosis and exchange during cell mating. Macronuclei, which contain only small, gene-sized DNA molecules, provide all of the nuclear RNA needed to run the cell. After cell mating the macronucleus is derived from a micronucleus, a derivation that includes excision of the genes from chromosomes and elimination of the remaining DNA. The eliminated DNA includes all of the repetitious sequences and approximately 95% of the unique sequences. We cloned large restriction fragments from the micronucleus that confer replication ability on a replication-deficient plasmid in Saccharomyces cerevisiae. Sequences that confer replication ability are called autonomously replicating sequences. The frequency and effectiveness of autonomously replicating sequences in micronuclear DNA are similar to those reported for DNAs of other organisms introduced into yeast cells. Of the 12 micronuclear fragments with autonomously replicating sequence activity, 9 also showed homology to macronuclear DNA, indicating that they contain a macronuclear gene sequence. We conclude from this that autonomously replicating sequence activity is nonrandomly distributed throughout micronuclear DNA and is preferentially associated with those regions of micronuclear DNA that contain genes.     

Regional organization of eukaryotic DNA sequences as studied by the formation of folded rings

The observed frequency of folded rings has been determined as a function of fragment length and degree of resection for DNA from mouse and Necturus. The thermal stability of the ring closure and the kinetics of ring formation have been studied. As seen in the case of Drosophila DNA, mouse and Necturus DNA display a decreasing frequency of folded rings as fragment length increases. We interpret this to mean that repetitious sequences of a given type are clustered into many thousands of characteristic regions, called g-regions. The present paper focuses on the interior organization of g-regions. Variations of two competing models may be entertained: “tandem repetition” and “intermittent repetition”. If the g-regions were composed of exact, tandemly-repeating sequences, all observations can be easily explained. In order to maintain the idea that the g-regions contain repetitious blocks located at regular, or irregular intervals, one must suppose that such repetitious blocks are long (>200 nucleotide pairs), not internally repetitious, and represent perhaps 80% of the nucleotides in the g-region. Such a sequence can be thought of as a fractional-tandem repeat. For example: HIJXXXABC … HIJXXXABC … HIJXXX, where the X's stand for nucleotides composing sequences that are unrelated to each other, and the letters (ABC … HIJ) represent nucleotides in the non-internally-repetitive repeating sequence. We feel that debate cart now be profitably devoted to the question of whether approximately 80 or 100% of the tandemly-repetitious unit is in fact tandem.     

Repeated DNA sequences found in the large spacer of Vicia faba rDNA

In the large spacer of the rDNA of Vicia faba, multiples of a 0.32 kilobasepair (kb) sequence reiterate to various degrees. We sequenced the repetitious region consisting of the repeating sequences and its flanking regions using two cloned plasmids, which contain V. faba rDNA segments encompassing the whole region of the large spacer. The repetitious region was found to consist of multiple complete copies and one truncated copy of a 325 bp repeat unit and to be flanked by direct repeat sequences of about 150 bp. The set of direct repeats located at either side of the repetitious region differed from each other with about 10% sequence heterogeneity. However, nucleotide sequences of the direct repeats were well conserved between the two clones examined. Southern blot hybridization indicated a widespread distribution within the whole V. faba genome of some related sequences with high homologies to the 325 bp repeat unit and to the direct repeats.     

Structure and genomic organization of proretrovirus-like elements partially eliminated from the somatic genome of Ascaris lumbricoides.

A clone containing a middle repetitive element next to satellite DNA has been isolated from a germ line genomic library of the chromatin eliminating nematode Ascaris lumbricoides var. suum. The structure of this element has been elucidated by comparison of several clones containing the element in different environments. It is flanked by 256-bp-long terminal repeats (LTRs) and has an internal region of approximately 7 kb. The nucleotide sequences of both the 5' and the 3' LTRs have been determined. The element has a strong structural similarity with retroviral proviruses and related mobile elements. It was therefore named 'Tas', for transposon-like element of Ascaris. Approximately 50 Tas copies are dispersed over approximately 20 different chromosomal sites. Their genomic distribution varies between individuals, indicating that Tas elements are mobile in the Ascaris genome. Two variant forms, Tas-1 and Tas-2, present in a ratio of approximately 2 to 1 in the germ line genome, have been characterized. They differ not only in their restriction pattern, but also in their elimination behaviour. While only about one-fourth of the Tas-1 elements are expelled from the somatic cell lineage, all Tas-2 copies are specifically eliminated and are thus confined to the germ line cells. We have demonstrated that a cloned representative of Tas-1 elements is expelled concomitantly with its flanking DNA sequences during the chromatin elimination process.     

Chromatin diminution in the copepod Mesocyclops edax: diminution of tandemly repeated DNA families from somatic cells.

Chromatin diminution, i.e., the loss of selected chromosomal regions during the differentiation of early embryonic cells into somatic cells, has been described in taxa as varied as ciliates, copepods, insects, nematodes, and hagfish. The nature of the eliminated DNA has been extensively studied in ciliate, nematode, and hagfish species. However, the small size of copepods, which makes it difficult to obtain enough DNA from early embryonic cells for cloning and sequencing, has limited such studies. Here, to identify the sequences eliminated from the somatic cells of a copepod species that undergoes chromatin diminution, we randomly amplified DNA fragments from germ line and somatic line cells of Mesocyclops edax, a freshwater cyclopoid copepod. Of 47 randomly amplified germ line clones, 45 (96%) contained short, tandemly repeated sequences composed of either 2 bp CA-repeats, 8 bp CAAATAGA-repeats, or 9 bp CAAATTAAA-repeats. In contrast, of 83 randomly amplified somatic line clones, only 47 (57%) contained such short, tandemly repeated sequences. As previously observed in some nematode species, our results therefore show that there is partial elimination of chromosomal regions containing (CAAATAGA and CAAATTAAA) repeated sequences during the chromatin diminution observed in the somatic cells of M. edax. We speculate that chromatin diminution might have evolved repeatedly by recruitment of RNAi-related mechanisms to eliminate nonfunctional tandemly repeated DNA sequences from the somatic genome of some species.     

LIA4 Encodes a Chromoshadow Domain Protein Required for Genomewide DNA Rearrangements in Tetrahymena thermophila

Extensive DNA elimination occurs as part of macronuclear differentiation during Tetrahymena sexual reproduction. The identification of sequences to excise is guided by a specialized RNA interference (RNAi) machinery that targets the methylation of histone H3 lysine 9 (K9) and K27 on chromatin associated with these internal eliminated sequences (IESs). This modified chromatin is reorganized into heterochromatic subnuclear foci, which is a hallmark of their subsequent elimination. Here, we demonstrate that Lia4, a chromoshadow domain-containing protein, is an essential component in this DNA elimination pathway. LIA4 knockout (ΔLIA4) lines fail to excise IESs from their developing somatic genome and arrest at a late stage of conjugation. Lia4 acts after RNAi-guided heterochromatin formation, as both H3K9 and H3K27 methylation are established. Nevertheless, without LIA4, these cells fail to form the heterochromatic foci associated with DNA rearrangement, and Lia4 accumulates in the foci, indicating that Lia4 plays a key role in their structure. These data indicate a critical role for Lia4 in organizing the nucleus during Tetrahymena macronuclear differentiation.     

De novo telomere addition to spacer sequences prior to their developmental degradation in Euplotes crassus

During sexual reproduction, Euplotes crassus precisely fragments its micronuclear chromosomes and synthesizes new telomeres onto the resulting DNA ends to generate functional macronuclear minichromosomes. In the micronuclear chromosomes, the macronuclear-destined sequences are typically separated from each other by spacer DNA segments, which are eliminated following chromosome fragmentation. Recently, in vivo chromosome fragmentation intermediates that had not yet undergone telomere addition have been characterized. The ends of both the macronuclear-destined and eliminated spacers were found to consist of six-base, 3′ overhangs. As this terminal structure on the macronuclear-destined sequences serves as the substrate for de novo telomere addition, we sought to determine if the spacer DNAs might also undergo telomere addition prior to their elimination. Using a polymerase chain reaction approach, we found that at least some spacer DNAs undergo de novo telomere addition. In contrast to macronuclear-destined sequences, heterogeneity could be observed in the position of telomeric repeat addition. The observation of spacer DNAs with telomeric repeats makes it unlikely that differential telomere addition is responsible for differentiating between retained and eliminated DNA. The heterogeneity in telomere addition sites for spacer DNA also resembles the situation found for telomeric repeat addition to macronuclear-destined sequences in other ciliate species.     

High-affinity microtubule protein-higher organism DNA complexes. Many-fold enrichment in repetitive mouse DNA sequences comprised of satellite DNAs

We have examined aspects of the interaction of cycled microtubule protein preparations with ³⁵S-labeled mouse DNA tracer in a competition system with unlabelled competitor E. coli or mouse DNA. The nitrocellulose filter binding assay was used to measure interaction by scintillation counting. DNA molecular weight affected the levels of filter retained ³⁵S-labelled mouse tracer DNA. Filter retention levels increased if ³⁵S-labelled mouse DNA tracer size was increased, and the filter binding level decreased if competitor DNA size was increased. There was a sizeable, reproducible difference in the ³⁵S-labelled mouse DNA tracer binding level of about 1% when E. coli or mouse DNA competitors were compared. Mouse DNA more effectively competed with ³⁵S-labelled mouse DNA for microtubule protein binding than did E. coli DNA, suggesting that a small class of higher-organism DNA sequences interacts very strongly with microtubule protein. From other studies we know this to be the MAP fraction (Marx, K.A. and Denial, T. (1984) in The Molecular Basis of Cancer (Rein, R., ed.), Alan R. Liss, New York, in the press; and Villasante, E., Corces, V.G., Manso-Martinez, R. and Avila, J. (1981) Nucleic Acids Res. 9, 895–908). We find that this difference in competitor DNA strength is qualitatively similar under high-stringency conditions (0.5 M NaCl, high competitor [DNA]) we developed for examining high-affinity complexes. Under high-stringency conditions we isolated 1.2% and 0.6% of ³⁵S-labelled mouse DNA at 4200 and 350 bp respective sizes as nitrocellulose filter bound DNA-protein complexes. At both molecular weights these high-affinity DNA sequences, isolated from the filters, were shown to be significantly enriched in repetitive DNA sequences by S₁ nuclease solution reassociation kinetics. The kinetics are consistent with about a 4-fold mouse satellite DNA enrichment as well as enrichment in other repetitious DNA sequence classes. The high molecular weight filter-bound DNA samples were sedimented to equilibrium in CsCl buoyant density gradients and found to contain primarily mouse satellite DNA density sequences (1.691 g/cm³) with some minor fractions at other density positions (1.670, 1.682, 1.705, 1.740, 1.760 g/cm³) similar to those observed by our laboratory in previous investigations of micrococcal nuclease-resistant chromatin (Marx, K.A. (1977) Biochem. Biophys. Res. Commun. 78, 777–784). That the high-affinity microtubule-bound DNA was some 3–5-fold enriched in mouse satellite sequences was demonstrated by its characteristic BstNI restriction enzyme cleavage pattern     

Evidence for transposition of dispersed repetitive DNA families in yeast.

Dispersed repetitive DNA sequences from yeast (Saccharomyces cerevisiae) nuclear DNA have been isolated as molecular hybrids in lambdagt. Related S. cerevisiae strains show marked alterations in the size of the restriction fragments containing these repetitive DNAs. "Ty1" is one such family of repeated sequences in yeast and consists of a 5.6 kilobase (kb) sequence including a noninverted 0.25 kb sequence of another repetitious family, "delta", on each end. There are about 35 copies of Ty1 and at least 100 copies of delta (not always associated with Ty1) in the haploid genome. A few Ty1 elements are tandem and/or circular, but most are disperse and show (along with delta) some sequence divergence between repeat units. Sequence alterations involving Ty1 elements have been found during the continual propagation of a single yeast clone over the course of a month. One region with a large number of delta sequences (SUP4) also shows a high frequency of sequence alterations when different strains are compared. One of the differences between two such strains involves the presence or absence of a Ty1 element. The novel joint is at one inverted pair of delta sequences.