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.     

Invention and Mystery in Hypotrich DNA1

ABSTRACT “The capacity to blunder slightly is the real marvel of DNA. Without this special attribute, we would still be anaerobic bacteria and there would be no music.” Lewis Thomas³ Hypotrichs have evolved extraordinary ways of organizing, manipulating, and replicating the DNA in their micronuclear and macronuclear genomes. Short macronuclear DNA molecules containing single genes are created by excision from chromosomes, accompanied by massive elimination of the germline DNA sequences between genes. Germline genes themselves are interrupted by multiple noncoding segments called internal eliminated segments, or IESs, that divide genes into multiple macronuclear-destined segments, or MDSs. The functional significance of this organization is unknown. Over evolutionary time IESs accumulate mutations rapidly are inserted into or excised from genes, and shift position along DNA molecules. MDSs are ligated to create functional genes when IESs are spliced out of micronuclear DNA during macronuclear development. MDSs in some germline genes are in scrambled disorder and become unscrambled in association with IES elimination. Replication of DNA in the macronucleus is accomplished by organization of replication enzymes and factors into a structure that sweeps through the macronucleus to replicate the many millions of gene-sized DNA molecules. The significance of many of the bizarre DNA phenomena in the evolutionary/functional success of hypotrichs is still unclear.     

Identification of an origin of bidirectional DNA replication in mammalian chromosomes

Mechanistically, an origin of bidirectional DNA replication (OBR) can be defined by the transition from discontinuous to continuous DNA synthesis that must occur on each template strand at the site where replication forks originate. This results from synthesis of Okazaki fragments predominantly on the retrograde arms of forks. We have identified these transitions at a specific site within a 0.45 kb sequence approximately 17 kb downstream from the 3' end of the dihydrofolate reductase gene in Chinese hamster ovary chromosomes. At least 80% of the replication forks in a 27 kb region emanated from this OBR. Thus, initiation of DNA replication in mammalian chromosomes uses the same replication fork mechanism previously described in a variety of prokaryotic and eukaryotic genomes, suggesting that mammalian chromosomes also utilize specific cis-acting sequences as origins of DNA replication.     

Developmentally programmed excision of internal DNA sequences in Paramecium aurelia

The development of a new somatic nucleus (macronucleus) during sexual reproduction of the ciliate Paramecium aurelia involves reproducible chromosomal rearrangements that affect the entire germline genome. Macronuclear development can be induced experimentally, which makes P. aurelia an attractive model for the study of the mechanism and the regulation of DNA rearrangements. Two major types of rearrangements have been identified: the fragmentation of the germline chromosomes, followed by the formation of the new macronuclear chromosome ends in association with imprecise DNA elimination, and the precise excision of internal eliminated sequences (IESs). All IESs identified so far are short, A/T rich and non-coding elements. They are flanked by a direct repeat of a 5'-TA-3' dinucleotide, a single copy of which remains at the macronuclear junction after excision. The number of these single-copy sequences has been estimated to be around 60,000 per haploid genome. This review focuses on the current knowledge about the genetic and epigenetic determinants of IES elimination in P. aurelia, the analysis of excision products, and the tightly regulated timing of excision throughout macronuclear development. Several models for the molecular mechanism of IES excision will be discussed in relation to those proposed for DNA elimination in other ciliates.     

snRNA and heterochromatin formation are involved in DNA excision during macronuclear development in stichotrichous ciliates

Several models for specific excision of micronucleus-specific DNA sequences during macronuclear development in ciliates exist. While the template-guided recombination model suggests recombination events resulting in specific DNA excision and reordering of macronucleus-destined sequences (MDS) guided by a template, there is evidence that an RNA interference-related mechanism is involved in DNA elimination in holotrichous ciliates. We describe that in the stichotrichous ciliate Stylonychia, snRNAs homologous to micronucleus-specific sequences are synthesized during macronuclear differentiation. Western and in situ analyses demonstrate that histone H3 becomes methylated at K9 de novo during macronuclear differentiation, and chromatin immunoprecipitation revealed that micronucleus-specific sequences are associated with methylated H3. To link both observations, expression of a PIWI homolog, member of the RNA-induced silencing complex, was silenced. In these cells, the methylated micronucleus-specific histone H3 variant "X" is still present in macronuclear anlagen and no K9 methylation of histone H3 is observed. We suggest that snRNA recruits chromatin-modifying enzymes to sequences to be excised. Based on our and earlier observations, we believe that this mechanism is not sufficient for specific excision of sequences and reordering of MDS in the developing macronucleus and propose a model for internal eliminated sequence excision and MDS reordering in stichotrichous ciliates.     

Tissue-specific heterogeneity in DNA replication patterns of human X chromosomes

Regional DNA replication kinetics in human X chromosomes have been analysed using BrdU-33258 Hoechst-Giemsa techniques in five cell types from human females: amniotic fluid cells, fetal and adult skin fibroblasts, and fetal and adult peripheral lymphocytes. In all cell types, the late-replicating X chromosome can be distinguished from its active, earlyreplicating homologue, and both the early and late X exhibit temporally and regionally characteristic internal sequences of DNA replication. The replication pattern of the early X in amniotic fluid cells and skin fibroblasts is similar to that of the early X in lymphocytes, although certain discrete regions are later-replicating in these monolayer tissue culture cells than are the corresponding regions in lymphocytes. However, DNA replication kinetics in late X chromosomes from amniotic fluid cells and skin fibroblasts are strikingly different from those observed in lymphocytes with respect both to the initiation and termination of DNA synthesis. The predominant late X pattern observed in 80–95% of lymphocytes, in which replication terminates in the long arm in bands Xq21 and Xq23, was never seen in amniotic fluid cells or skin fibroblasts. Instead, in these cell types, bands Xq25 and Xq27 are the last to complete DNA synthesis, while bands Xq21 and Xq23 are earlier-replicating; this pattern is similar to the alternative replication sequence observed in 5–20% of lymphocyte late X chromosomes. This replication sequence heterogeneity is consistent with the existence of tissue-specific influences on the control of DNA replication in human X chromosomes.     

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.     

Sequential excision of internal eliminated DNA sequences in the differentiating macronucleus of the hypotrichous ciliate Stylonychia lemnae.

Elimination of internal eliminated sequences (IES) during macronuclear development of the hypotrichous ciliate Stylonychia lemnae was analyzed in one cluster of macronuclear precursor DNA sequences. The results indicate that IES elimination is a highly ordered process, it starts very early during macronuclear development and has only finished immediately before DNA fragmentation takes place. It occurs in distinct steps and the IES are eliminated in a specific order, where a defined IES is only removed after complete elimination of other IES. Transfection experiments clearly demonstrate that the structure of the IES itself is not sufficient for its correct excision but other cis-acting sequences or additional structural requirements are needed for IES elimination.     

Developmentally programmed excision of internal DNA sequences in Paramecium aurelia

The development of a new somatic nucleus (macronucleus) during sexual reproduction of the ciliate Paramecium aurelia involves reproducible chromosomal rearrangements that affect the entire germline genome. Macronuclear development can be induced experimentally, which makes P. aurelia an attractive model for the study of the mechanism and the regulation of DNA rearrangements. Two major types of rearrangements have been identified: the fragmentation of the germline chromosomes, followed by the formation of the new macronuclear chromosome ends in association with imprecise DNA elimination, and the precise excision of internal eliminated sequences (IESs). All IESs identified so far are short, A/T rich and non-coding elements. They are flanked by a direct repeat of a 5’-TA-3’ dinucleotide, a single copy of which remains at the macronuclear junction after excision. The number of these single-copy sequences has been estimated to be around 60 000 per haploid genome. This review focuses on the current knowledge about the genetic and epigenetic determinants of IES elimination in P. aurelia, the analysis of excision products, and the tightly regulated timing of excision throughout macronuclear development. Several models for the molecular mechanism of IES excision will be discussed in relation to those proposed for DNA elimination in other ciliates.     

Multiple pathways cooperate to facilitate DNA replication fork progression through alkylated DNA

Eukaryotic genomes are especially vulnerable to DNA damage during the S phase of the cell cycle, when chromosomes must be duplicated. The stability of DNA replication forks is critical to achieve faithful chromosome replication and is severely compromised when forks encounter DNA lesions. To maintain genome integrity, replication forks need to be protected by the S-phase checkpoint and DNA insults must be repaired. Different pathways help to repair or tolerate the lesions in the DNA, but their contribution to the progression of replication forks through damaged DNA is not well known. Here we show in budding yeast that, when the DNA template is damaged with the alkylating agent methyl methanesulfonate (MMS), base excision repair, homologous recombination and DNA damage tolerance pathways, together with a functional S-phase checkpoint, are essential for the efficient progression of DNA replication forks and the maintenance of cell survival. In the absence of base excision repair, replication forks stall reversibly in cells exposed to MMS. This repair reaction is necessary to eliminate the lesions that impede fork progression and has to be coordinated with recombination and damage tolerance activities to avoid fork collapse and allow forks to resume and complete chromosome replication.     

Mapping of the pattern of DNA replication in polytene chromosome from Chironomus thummi using monoclonal anti-bromodeoxyuridine antibodies

We present results from a nonautoradiographic study of DNA replication in polytene chromosomes from dipteran larvae. Monoclonal antibodies with specificity for 5-bromodeoxyuridine (BrdUrd) were used to localize by indirect immunofluorescence the sites of BrdUrd incorporation and to follow the dynamics of DNA synthesis in salivary gland cells of 4th instar Chironomus thummi larvae. This technique presents numerous advantages over autoradiographic procedures and allows mapping of DNA synthesis patterns at the level of resolution of one chromosomal band. Several replication patterns were observed, classified according to characteristic features, and tentatively assigned to specific periods of the S-phase. In early S-phase, DNA synthesis is first detectable in puffs and interbands, later in bands. Most chromosomal bands appear to initiate DNA synthesis synchronously; however, in bands within centromeric and heterochromatic regions the start of synthesis is delayed. At mid S-phase, all the bands show uniform staining. Subsequent staining patterns are increasingly differential with the bands displaying characteristic fluorescence intensities. As replication progresses through the late S-phase period, the chromosomes show a decreasing number of fluorescent bands. The last bands to terminate replication are located in centromeric and heterochromatic DNA-rich regions and a few bands of low DNA content in region IIAa-c.     

Differential incorporation of halogenated deoxyuridines during UV-induced DNA repair synthesis in human cells

Double labeling of interphase and metaphase chromosomes by 5-chlorodeoxyuridine (CldU) and 5-iododeoxyuridine (IdU) has been used in studies of the dynamics of DNA replication. Here, we have used this approach and confocal microscopy to analyze sites of DNA repair synthesis during nucleotide excision repair (NER) in quiescent human fibroblasts. Surprisingly, we have found that when both precursors are added at the same time to UV-irradiated cells they label different sites in the nucleus. In contrast, even very short periods of simultaneous IdU+CldU labeling of S-phase cells produced mostly overlapped IdU and CldU replication foci. The differential labeling of repair sites might be due to compartmentalization of I-dUTP and Cl-dUTP pools, or to differential utilization of these thymidine analogs by DNA polymerases delta and epsilon (Poldelta and Polepsilon). To explore the latter possibility we used purified mammalian polymerases to find that I-dUTP is efficiently utilized by both Poldelta and Polepsilon. However, we found that the UV-induced incorporation of IdU was more strongly stimulated by treatment of cells with hydroxyurea than was incorporation of CldU. This indicates that there may be distinct IdU and CldU-derived nucleotide pools differentially affected by inhibition of the ribonucleotide reductase pathway of dNTP synthesis and that is consistent with the view that differential incorporation of IdU and CldU during NER may be caused by compartmentalization of IdU- and CldU-derived nucleotide pools.     

A model for replication of the ends of linear chromosomes

Linear chromosomes possessing internal repeats of their terminal sequences can form intramolecular crossed-strand exchanges that allow replication of the chromosome ends. Evidence is discussed that such a mechanism may be utilized during replication of herpes simplex virus DNA and during replication of macronuclear DNA from the hypotrichous ciliate Oxytricha.     

Role of histone deacetylation in developmentally programmed DNA rearrangements in Tetrahymena thermophila

In Tetrahymena, as in other ciliates, development of the somatic macronucleus during conjugation involves extensive and reproducible rearrangements of the germ line genome, including chromosome fragmentation and excision of internal eliminated sequences (IESs). The molecular mechanisms controlling these events are poorly understood. To investigate the role that histone acetylation may play in the regulation of these processes, we treated Tetrahymena cells during conjugation with the histone deacetylase inhibitor trichostatin A (TSA). We show that TSA treatment induces developmental arrests in the early stages of conjugation but does not significantly affect the progression of conjugation once the mitotic divisions of the zygotic nucleus have occurred. Progeny produced from TSA-treated cells were examined for effects on IES excision and chromosome breakage. We found that TSA treatment caused partial inhibition of excision of five out of the six IESs analyzed but did not affect chromosome breakage at four different sites. TSA treatment greatly delayed in some cells and inhibited in most the excision events in the developing macronucleus. It also led to loss of the specialized subnuclear localization of the chromodomain protein Pdd1p that is normally associated with DNA elimination. We propose a model in which underacetylated nucleosomes mark germ line-limited sequences for excision.     

DNA rearrangements and mating-type determination in Paramecium tetraurelia

Ciliated protozoa have separate germline and somatic nuclei, yet unlike larger organisms, both nuclei reside in the same cytoplasm. The micronuclei contain the germline and the macronucleus is the somatic nucleus. Thousands of DNA elements are normally removed from the micronuclear genome as it forms a new macronucleus during each sexual cycle. A recent study directly links the excision of these internal eliminated sequences (IESs) to mating type determination by showing that a pleiotropic mutation affecting mating type also prevents the excision of an IES from a surface protein gene⁽¹⁾. Remarkably, once the IES is present in the old macronucleus it prevents excision of that specific IES during formation of the next macronucleus.     

Micronuclear genome organization in Euplotes crassus: a transposonlike element is removed during macronuclear development.

After mating, hypotrichous ciliated protozoa transform a set of their micronuclear chromosomes into thousands of short, linear DNA molecules that form the macronuclear genome. To examine micronuclear genome organization in the hypotrich Euplotes crassus, we have analyzed two cloned segments of micronuclear DNA as well as the macronuclear DNA molecules that are derived from them. E. crassus was found to display a number of features characteristic of other hypotrich genomes, including (i) clustering and close spacing of the precursors of macronuclear DNA molecules, (ii) the frequent occurrence of internal eliminated sequences within macronuclear precursors, (iii) overlapping macronuclear precursors, (iv) lack of telomeric repeats at the ends of macronuclear precursors, and (v) alternative processing of the micronuclear chromosome to yield multiple macronuclear DNA molecules. In addition, a moderately repetitive, transposonlike element that interrupts the precursors of two macronuclear DNA molecules has been identified and characterized. This transposonlike element, designated Tec1, is shown to be reproducibly removed from one of the macronuclear precursors during independent episodes of macronuclear development.