Extrachromosomal DNA and its activity in RNA synthesis in oogonia and oocytes in the pupal ovary of Creophilus maxillosus (Staphylinidae, Coleoptera-Polyphaga)

The participation of extrachromosomal DNA (extra DNA) in RNA synthesis in the nuclei of terminal oogonial cells and oocytes in the pupal ovary of Creophilus maxillosus (Staphylinidae, Coleoptera) was examined by autoradiography. It was found that extra DNA in the nuclei of terminal oogonial cells, although predominantly in a condensed and heterochromatic state, produces numerous nucleoli and incorporates 3H-uridine during the interphases between successive differential divisions. Moreover, it was shown that extra DNA is active in RNA synthesis at the same stage of pupal development in which it is synthesized and accumulated, i.e. in the nuclei of terminal oogonial cells. As soon as the oocyte forms RNA synthesis ceases in the extrachromosomal DNA body cells showed that nucleolar material does not disappear during division but remains, at least partly, connected with the extra DNA body.     

Extrachromosomal DNA and the origin of oocytes in the telotrophic-meroistic ovary ofCreophilus maxillosus (L.) (Staphylinidae,Coleoptera-Polyphaga)

Summary The development of the telotrophic ovary in the Staphylinid beetle,Creophilus maxillosus was examined. Cells, termed chordoblasts were identified in the germarium of 1-day-old pupae. Each of the chordoblasts undergoes a series of synchronous mitoses. Owing to the precise control of the cleavage plane, which is vertical to the long axis of the ovariole, each of the chordoblasts gives rise to a linear chain of sibling chordocytes. Extra DNA synthesis within each sibling string is usually limited to the most posterior chordocyte only, this being an oocyte progenitor.Divisions of the oocyte progenitor are differential mitoses in which the extra DNA material is transported preferentially towards the posterior pole of the spindle. As extra DNA synthesis and preferential segregation of this material result in gradual increase of this DNA in the nuclei of oocyte progenitors, cytokinesis of these cells becomes highly unequal, the larger of the two cells produced at each differential mitosis being as a rule the posterior cell, i.e. the oocyte progenitor of the next cell generation. As a resul of the series of differential mitoses each chordoblast gives rise to a number of nurse cells and only one definitive oocyte.It is suggested that somatic prefollicular tissue plays a decisive role in oocyte determination in the Coleopteran telotrophic ovary.This word was supported in part under Contract DPKBN/52/76-II. 1. 3. 10. with the Polish Academy of Science     

Germ-cell cluster formation in the telotrophic meroistic ovary of <Emphasis Type="Italic">Tribolium castaneum</Emphasis> (Coleoptera,Polyphaga, Tenebrionidae) and its implication on insect phylogeny

Tribolium castaneum has telotrophic meroistic ovarioles of the Polyphaga type. During larval stages, germ cells multiply in a first mitotic cycle forming many small, irregularly branched germ-cell clusters which colonize between the anterior and posterior somatic tissues in each ovariole. Because germ-cell multiplication is accompanied by cluster splitting, we assume a very low number of germ cells per ovariole at the beginning of ovariole development. In the late larval and early pupal stages, we found programmed cell death of germ-cell clusters that are located in anterior and middle regions of the ovarioles. Only those clusters survive that rest on posterior somatic tissue. The germ cells that are in direct contact with posterior somatic cells transform into morphologically distinct pro-oocytes. Intercellular bridges interconnecting pro-oocytes are located posteriorly and are filled with fusomes that regularly fuse to form polyfusomes. Intercellular bridges connecting pro-oocytes to pro-nurse cells are always positioned anteriorly and contain small fusomal plugs. During pupal stages, a second wave of metasynchronous mitoses is initiated by the pro-oocytes, leading to linear subclusters with few bifurcations. We assume that the pro-oocytes together with posterior somatic cells build the center of determination and differentiation of germ cells throughout the larval, pupal, and adult stages. The early developmental pattern of germ-cell multiplication is highly similar to the events known from the telotrophic ovary of the Sialis type. We conclude that among the common ancestors of Neuropterida and Coleoptera, a telotrophic meroistic ovary of the Sialis type evolved, which still exists in Sialidae, Raphidioptera, and a myxophagan Coleoptera family, the Hydroscaphidae. Consequently, the telotrophic ovary of the Polyphaga type evolved from the Sialis type. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Sequence and structure of the extrachromosomal palindrome encoding the ribosomal RNA genes in Dictyostelium

Ribosomal RNAs (rRNAs) are encoded by multicopy families of identical genes. In Dictyostelium and other protists, the rDNA is carried on extrachromosomal palindromic elements that comprise up to 20% of the nuclear DNA. We present the sequence of the 88 kb Dictyostelium rDNA element, noting that the rRNA genes are likely to be the only transcribed regions. By interrogating a library of ordered YAC clones, we provide evidence for a chromosomal copy of the rDNA on chromosome 4. This locus may provide master copies for the stable transmission of the extrachromosomal elements. The extrachromosomal elements were also found to form chromosome-sized clusters of DNA within nuclei of nocodazole-treated cells arrested in mitosis. These clusters resemble true chromosomes and may allow the efficient segregation of the rDNA during mitosis. These rDNA clusters may also explain the cytological observations of a seventh chromosome in this organism.     

Single extrachromosomal ribosomal rna gene copies are synthesized during amplification of the rDNA in tetrahymena

A novel form of extrachromosomal rDNA has been identified in conjugating Tetrahymena cells. This rDNA consists of 11 kb linear double-stranded DNA molecules, each containing a single rRNA gene copy. The DNA sequence, tandemly repeated CCCCAA (Blackburn and Gall 1978) found at the termini of extrachromosomal palindromic rDNA (the macronuclear form found in vegetatively growing cells), is also present at the corresponding terminus of the 11 kb rDNA. The other end of this molecule has an extra 0.3 kb segment of DNA covalently attached to the DNA region corresponding to the center of the palindromic rDNA. The kinetics of appearance and synthesis of the 11 kb rDNA early in macronuclear development are consistent with its being an intermediate in rDNA amplification.     

Extrachromosomal rDNA amplification in the oocytes of Polystoechotes punctatus (Fabricius) (Insecta-Neuroptera-Polystoechotidae)

The ovary of Polystoechotes punctatus consists of several ovarioles of meroistic-polytrophic type. Histological, histochemical and ultrastructural studies revealed that the extrachromosomal amplification of rDNA takes place in the oocyte nucleus. Prior to previtellogenic growth the oocyte nucleus contains the chromosomes of meiotic prophase and a condensed extra DNA body. Initial split of extrachromosomal DNA material into several fragments coincides with the appearance of a spherical, fine granular body (referred to as primary nucleolus). Its gradual fragmentation accompanied by further dispersion of amplified DNA results in the formation of a growing number of multiple nucleoli. Until mid previtellogenesis each multiple nucleolus contains detectable amount of rDNA. In the advanced stages of previtellogenesis rDNA can hardly be visualized within the multiple nucleoli, while chromosomes form a few dense aggregates randomly disposed in the karyoplasm. At the onset of vitellogenesis the chromosomes assemble to form a karyosome. In its close vicinity DNA-positive material reaggregates. Multiple nucleoli are either found on the periphery of this aggregation or merge within it. At the final stages of vitellogenesis the number of multiple nucleoli significantly decreases.     

Early development of the kelp fly,Coelopa frigida (Diptera). II. Morphology of cleavage and blastoderm formation

Cleavage and blastoderm formation in Coelopa frigida are extremely rapid developmental processes. In short (6–7 minutes) successive cell cycles, nuclei multiply and spread out through the egg. The movement seems to be aided by endoplasmic vesicles and cisternae which are in direct contact with the nuclear membrane. The first cells to separate from the egg plasmodium in early superficial cleavage stages are the pole cells. Precursor material from multivesicular bodies forms the pole cell membranes. The primary nuclei from the posterior pole region are removed from the blastoderm by the pole cell segregation. Blastoderm nuclei from the regions adjacent to the posterior pole migrate into the residual periplasm after pole cell segregation has been completed and constitute the blastoderm nuclei in that region of the egg. Nucleoli are not revealed during internal cleavage. They appear in pole cells shortly after their segregation. The generation time of the blastoderm nuclei increases after the twelfth cleavage. Concurrently, nucleoli form in the blastoderm nuclei and permanent cell membranes separate individual blastoderm cells. After blastoderm cells have been separated from each other, they remain in contact with the interior yolk sac by means of cytoplasmic canals. This contact is maintained at least during the early phases of blastokinesis. Observations on nuclear migration and rapid membrane formation are discussed as examples of protein assembly from subunits as an alternative to de novo protein synthesis in early stages of development.     

Solitary and synaptonemal complex-associated recombination nodules in pro-nurse cells during oogenesis in Drosophila melanogaster

An oocyte in Drosophila melanogaster originates from 1 of 16 cells comprising an ovarial syncytium. The two pro-oocytes proceed into the pachytene stage of meiosis, but only one develops further into a mature oocyte while the other reverts to a nurse cell. It is known that pro-nurse cells also enter meiosis, as they contain incomplete synaptonemal complexes (SCs). We now show that these cells also harbour recombination nodules (RNs). In cells that only occasionally contain SC segments, the RNs are typically not located close to distinct tripartite SC structures. Instead, these RNs are frequently associated with a spherical body of amorphous material and two to three, more or less parallel fibres, possibly representing SC material. The significance of the solitary RNs is discussed in relation to the present knowledge of the assembly and disassembly of the SC.     

Gene amplification in the oocytes of dytiscid water beetles

A conspicuous mass of extrachromosomal DNA (Giardina's body) is found in oogonia and oocytes of Dytiscid water beetles. Since in older oocytes this DNA is associated with numerous nucleoli, it seemed probable that the ovary might contain extra copies of the genes for ribosomal RNA (rRNA). This hypothesis has been confirmed by centrifugation and molecular hybridization studies. —In Dytiscus marginalis and Colymbetes fuscus a high density satellite DNA is found in somatic cells and in sperm. Hybridization experiments show that all of the rDNA, i.e., those sequences complementary to rRNA, are located in this satellite, although quantitatively they make up only a small fraction of the satellite. In both species the DNA isolated from ovariole tips is enriched with respect to the satellite. A parallel enrichment of the rDNA has been shown in ovariole tips of Colymbetes, but for technical reasons has not been examined in Dytiscus. —The following model is proposed. In somatic cells and sperm the rDNA is part of an extensive region of high density DNA in one or more chromosomes. In oogonia and oocytes the entire high density region is replicated extrachromosomally and appears cytologically as Giardina's body.     

Photoreversible inhibition by ultraviolet light of germ line development in Smittia sp. (Chironomidae, Diptera)

Pole cell formation in embryos of the parthenogenetic midge, Smittia sp., can be delayed or inhibited by irradiation of the posterior egg pole with ultraviolet light (uv). This leaves the schedule of nuclear divisions and chromosome eliminations virtually unaffected. However, uv irradition delays the precocious migration to the posterior pole of one nucleus, which normally becomes included in the first pole cell. This effect is photoreversible, i.e., mitigated by application of blue light after uv. Photoreversibility indicates that a nucleic acid component is involved as an effective target. During normal development of Smittia a number of chromosomes are eliminated during mitosis V, not only from somatic nuclei but also in the germ line. In the latter, this mitosis takes place during the first gonial division in the larva. After uv irradiation, the first pole cell nucleus has undergone supernumerary mitoses before pole cell formation and, as a result, is driven into mitosis V precociously as the pole cell divides. This is frequently associated with chromosome elimination from pole cells, which in turn is correlated with subsequent disappearance of already formed pole cells. Adults derived from embryos without pole cells do not form ovaries. Pole cell formation, pole cell preservation, and ovary development are separately inhibited by uv, and inhibition of each step is photoreversible. The results are discussed in the context of germ cell determination, protection against chromosome elimination, and the role of chromosomes limited to the germ line.     

Developmental analysis of grandchildless (gs(1)N441) mutation in Drosophila melanogaster; abnormal formation of pole cells

A detailed examination of the developmental features of abnormal formation of pole cells and a functional analysis of the germ plasma of gs(1)N441 embryos were carried out. The germ plasma is morphologically normal. Embryos in which cleavage nuclei show retarded migration to the posterior pole do not form pole cells. Pole cells, following formation, are abnormally segregated and then intermingled between the blastoderm cell layer but retaining normal morphology and differentiating into functional germ cells. The results of cytoplasmic transplantation experiments indicate the autonomous segregation ability of the mutant polar plasma to form pole cells to possibly be affected.     

Structure of the germinal vesicle during oogenesis in leech Glossiphonia heteroclita (Annelida, Hirudinea, Rhynchobdellida)

Oogenesis in the glossiphoniid leech Glossiphonia heteroclita (Hirudinea, Rhynchobdellida) is nutrimental, i.e., the growing oocyte is supported by specialized germline cells, the nurse cells. The main function of the nurse cells is to provide oocytes with cell organelles and RNAs (mainly rRNA). However, in studied leech species, irrespective of the nutrimental mode of oogenesis, the germinal vesicle (GV = oocyte nucleus) seems to be very active in rRNA production. As shown in the present study, during early previtellogenesis in the GV the meiotic chromosomes and prominent primary nucleoli occur. In late previtellogenesis the chromosomes condense and occupy a limited space of nucleoplasm in close vicinity to primary nucleolus, forming a karyosome. At the onset of vitellogenesis several prominent extrachromosomal DNA bodies appear in close association with the karyosome. At the same time, the primary nucleolus is no longer visible in the GV. As vitellogenesis proceeds the extrachromosomal DNA bodies undergo fragmentation and numerous spherical, RNA- and AgNOR-positive inclusions occur in the nucleoplasm. They are regarded as multiple nucleoli. Finally, in late oogenesis numerous accessory nuclei are formed in close proximity to the nuclear envelope. They usually contain one dense body, morphologically similar to multiple nucleoli. The amplification of rDNA genes, the occurrence of extrachromosomal DNA bodies, as well as the presence of multiple nucleoli and accessory nuclei are described for the first time in the phylum Annelida.     

Extrachromosomal circular nuclear rDNA in Euglena gracilis.

The presence of extrachromosomal nuclear ribosomal DNA (rDNA) in the unicellular alga Euglena gracilis has been established. This rDNA is circular. Each circle is 3.8 micron long and contains one rDNA unit. Oligomers are rare. Extrachromosomal rDNA is present in large amounts during the exponential phase of growth and appears less abundant during the stationary phase. It was found in all wild-type and mutant strains of Euglena examined. Our estimations suggest that rDNA in Euglena is mainly extrachromosomal. Research of extrachromosomal rDNA in spinach and Petunia was negative.     

Rapid de novo centromere formation occurs independently of heterochromatin protein 1 in C. elegans embryos

DNA injected into the Caenorhabditis elegans germline forms extrachromosomal arrays that segregate during cell division [1, 2]. The mechanisms underlying array formation and segregation are not known. Here, we show that extrachromosomal arrays form de novo centromeres at high frequency, providing unique access to a process that occurs with extremely low frequency in other systems [3-8]. De novo centromerized arrays recruit centromeric chromatin and kinetochore proteins and autonomously segregate on the spindle. Live imaging following DNA injection revealed that arrays form after oocyte fertilization via homologous recombination and nonhomologous end-joining. Individual arrays gradually transition from passive inheritance to active segregation during the early embryonic divisions. The heterochromatin protein 1 (HP1) family proteins HPL-1 and HPL-2 are dispensable for de novo centromerization even though arrays become strongly enriched for the heterochromatin-associated H3K9me3 modification over time. Partial inhibition of HP1 family proteins accelerates the acquisition of segregation competence. In addition to reporting the first direct visualization of new centromere formation in living cells, these findings reveal that naked DNA rapidly builds de novo centromeres in C. elegans embryos in an HP1-independent manner and suggest that, rather than being a prerequisite, HP1-dependent heterochromatin antagonizes de novo centromerization.     

Amplified ribosomal DNA in meiotic prophase oocyte nuclei of acipenserid fishes

Summary In situ hybridization has been performed in sections through ovaries ofAcipenser ruthenus andAcipenser güldenstädti in order to detect the rDNA sequences. Hybridization resulted in specific labelling of the caps of extrachromosomal DNA present in pachytene oocyte nuclei and of the chromatin granules distributed beneath the nuclear envelope in early diplotene nuclei. In the same sections, the nuclei of all ovarian cells in both species (oogonia, leptotene, and zygotene stage oocytes, follicular cells, connective tissue cells) showed a very low, but similar labelling.Amplification of genes for rRNA thus occurs at the pachytene stage in early oogenesis ofAcipenseridae. No rDNA amplification could be detected in the previous stages.     

Germ cell cluster formation and ovariole structure in viviparous and oviparous generations of the aphid Stomaphis quercus

The developing ovaries of S. quercus contain a limited number of oogonial cells which undergo a series of incomplete mitotic divisions resulting in the formation of clusters of cystocytes. Ovaries of viviparous generations contain 6 to 9 clusters, containing 32 cystocytes each, whereas ovaries of oviparous generations contain 5 clusters containing 45-60 cystocytes. During further development, clusters become surrounded by a single layer of follicular cells, and within each cluster the cystocytes differentiate into oocytes and trophocytes (nurse cells). Concurrently, cysts transform into ovarioles. The anterior part of the ovariole containing the trophocytes becomes the tropharium, whereas its posterior part containing oocytes transforms into the vitellarium. The vitellaria of viviparous females are composed of one or two oocytes, which develop until previtellogenesis. The nuclei of previtellogenic oocytes enter cycles of mitotic divisions which lead to the formation of the embryo. Ovarioles of oviparous females contain a single oocyte which develops through three stages: previtellogenesis, vitellogenesis and choriogenesis. The ovaries are accompanied by large cells termed bacteriocytes which harbor endosymbiotic microorganisms.     

Absence of rDNA amplification in the uninucleolate oocyte of the cockroach Blattella germanica (Oorthoptera: Blattidae)

Amplification of the genes coding for ribosomal RNA oocurs in the oocytes of a wide variety of organisms. In oocytes of various species of crickets (Orthoptera: Gryllidae) the amplified DNA is contained in a large extrachromosomal DNA body. Multiple nucleoli form about the periphery of the DNA body during the diplotene stage of meiosis I. In contrast to the general pattern of orthopteran oocytes, oocytes of the cockroach Blattella germanica demonstrate a single large nucleolus instead of many nucleoli. In order to determine whether the genes coding for rRNA are amplified in the oocytes of B. germanica, the relative amount of rDNA in oocytes was compared with the rDNA content of spermatocytes and somatic cells. An extrachromosomal DNA body similar to that present in crickets is not present in B. germanica. A satellite DNA band which contains nucleotide sequences complementary to rRNA accounts for approximately 3-5% of the total DNA in somatic and in male and female gametogenic tissues. Female cells contain approximately twice as much rDNA as do male cells. An XX-XO sex-determining mechanism is operative in B. germanica. In situ hybridization with rRNA indicates that the nucleolar organizer is located on one end of the X chromosome and that oocytes do not contain more than twice the amount of rDNA found in spermato cytes. The data indicate that rDNA is not amplified in the uninucleolate oocyte of B germanica.     

Chromosome age and segregation during sporulation of Bacillus megaterium.

The effect of chromosome age on segregation during sporulation was investigated. Vegetative cells of Bacillus megaterium were labeled with [Me-3H]thymine and then were grown at 30 degrees C in nonradioactive medium for various times before being allowed to sporulate. The ratio of the amount of label in sporal DNA to that in sporangial DNA, obtained after minor correction for the sporulation frequency, remained essentially constant as the postlabeling growth period was increased from one to seven generations. The spores were preferentially located at the older poles of sporangia, i.e. the poles formed by divisions occurring prior to those forming the sporangia. Therefore, it seems that old (labeled) chromosomes segregate randomly with respect to both the morphological and genealogical polarities of sporangia. Examination of total cell lysates by dye-buoyant density gradient centrifugation revealed the presence of covalently closed circular DNA from cells grown at 37 degrees C, but none was obtained from cells grown at 30 degrees C. Thus, possible interference by large amounts of extrachromosomal DNA in the determination of the chromosomal segregation pattern is unlikely.