Under-replication of intron+ rDNA cistrons in polyploid nurse cell nuclei of Calliphora erythrocephala

We have examined the possibility that the intron-containing (intron⁺) rDNA cistrons of Dipteran flies are active in the germ-line derived polyploid nurse cell nuclei of the ovarian follicles. Using the organism, Calliphora erythrocephala, we describe here a procedure which yields very pure nurse cell nuclei and compare the intron-free (intron^–) and intron⁺ rDNA cistron contents of nurse cell nuclei prepared by this procedure to those of 2–18 h embryo nuclei and 3 day pupal nuclei. DNA from three preparations of each nuclear type was examined and the intron^– and intron⁺ cistron contents quantitated using a Southern transfer procedure. The number of intron^– and intron⁺ rDNA cistrons per haploid genome in the presumed diploid 2–18 h embryo DNA was first established, and then the intron^– and intron⁺ cistron contents of nurse cell nuclear DNA and 3 day pupal DNA were determined relative to these values.The intron^– cistron content of nurse cell nuclear DNA was indistinguishable from that of embryonic DNA but the intron⁺ cistrons showed an 8-fold under-replication relative to the presumed diploid DNA. A slight under-representation of the intron^– cistrons and 3-fold under-replication of the intron⁺ cistrons were demonstrated for 3 day pupal DNA. These findings strongly suggest that intron⁺ rDNA cistrons are non-functional in nurse cell nuclei and substantiate the generality of this implication for the whole organism during early pupal life.     

Organization and differential staining of chromosomes from the endomitotic nucleus of trophocytes Calliphora erythrocephala (Diptera: Calliphoridae)

The structure of primary polytene chromosomes and general architecture of nurse cell nuclei was studied in Calliphora erythrocephala using various methods of differential chromosome banding(G-, R-, C-banding; Ag- and DAPI staining), chromospecific DNA probes and fluorescence in situ hybridization. This analysis revealed differential compaction of particular chromosome regions. The localization of material of polytene chromosome 6 is retained after its rearrangement and the formation of the internal reticular structure of the nucleus. Polytene chromosomes of ovarian nurse cells were shown to have blocks of dense compact material; some of them were more intensely stained by AgNO3. The dynamics of the nucleolus formation was traces at all stages of chromosome polytenization in the C. erythrocephala nurse cells.     

Both Nucleolar Organizers Are Replicated in Dipteran Polyploid Tissues: A Study at the Level of Individual Nuclei

Working with the Dipteran Calliphora erythrocephala, we have tested the hypothesis that only one nucleolar organizer region (NO) is replicated during polyploidization. NO replication was examined in two very different highly polyploid nuclear types: salivary gland nuclei and nurse cell nuclei. Two strains of the organism containing NO regions with highly diagnostic nontranscribed spacer (NTS) polymorphisms were prepared and reciprocal single pair-matings between members of the strains were performed. The representation of the two distinguishable NOs in diploid and polyploid DNAs of individual F1 progeny from each cross was then examined. DNA from a total polyploid nuclear DNA preparation and from individual polyploid nuclei of both tissue types was analyzed. Our results show conclusively that both genomic NOs are replicated in individual polyploid nuclei of both types. Further, evidence for variation in the relative replication of cistrons from the two NOs by individual nuclei was obtained. The cistron types present in the NOs of both strains showed differential replication upon polyploidization. In general, the patterns of differential cistron replication seen in salivary gland and nurse cell nuclei were similar.     

Effect of diet on the neuroendocrine system and egg development in the sheep blowfly, Lucilia sericata

ABSTRACT. A light microscope study of the endocrine and ovarian systems of Lucilia sericata under two diets revealed that in young females fed on sugar and water, medial neurosecretory cells (MNC) synthesized and stored neurosecretory material (NSM) as the flies matured. The MNC remained filled with NSM as long as the diet was maintained. Following a small increase immediately after emergence, the size of the corpora allata (CA) showed little further change, and the nuclei of nurse cells remained small. However, rapid changes occurred in these tissues soon after a meat meal: NSM was discharged from the MNC, and the CA increased in size. These changes were at a maximum 20 h after a meat meal. 4h later, vitellogenesis was well established and the nurse cell nuclei had increased in size 20-fold. Growth of the nurse cell nuclei continued until approximately 6 h before the completion of vitellogenesis when they are resorbed. Oögenesis took about 48 h at 25°C. When 100 μg of each of three different juvenoids were applied topically to different sugar-fed flies, the nuclei of both MNC and nurse cells became enlarged, whereas the CA were somewhat reduced in size. The relationship between protein ingestion and oogenesis is discussed, and the results obtained with L. sericata are compared with those of other species, especially the blowfly Calliphora erythrocephala.     

Intranuclear dynamics of chromosome 6 in nurse cells of Calliphora erythrocephala Mg. (Diptera: Calliphoridae)

Intranuclear dynamics of chromosome 6 in nurse cell nuclei of Calliphora erythrocephala Mg. (Diptera: Calliphoridae) was studied. The 3D FISH method was used for the first time to study chromosome territories in highly polyploid nuclei whose chromosomes undergo morphological changes. A considerable change in the intranuclear location of chromosome 6 and a morphological alteration of the chromosome territory in the course of chromatin polytenization were revealed.     

A cytophotometric analysis of the structure of hypothalamic cell populations in the frog,Rana temporaria (L.), with special reference to seasonal changes in chromatin status

Summary We used cytophotometry after the Feulgen reaction and UV cytophotometry to measure the DNA content of quiescent cells of the hypothalamic preoptic region (HPR) of adult and juvenile frogs (Rana temporaria) that had been caught in their natural habitat in winter, spring and summer. The histone-to-DNA ratio in cell nuclei was cytophotometrically determined using a combined Feulgen, heparine and alcian-blue staining procedure. The vast majority of HPR cells studied had nuclei with a diploid DNA content. However, we observed great variability in the Feulgen-DNA content of the HPR cell population, which was not detected in the diploid standard (hepatocytes). This heterogeneity in the diploid sample of the HPR cell populations was always greater in prespawning frogs and may have been due to differences in the chromatin arrangement in nuclei. About 1% of cells had a DNA content either ranging between diploid and tetraploid levels (H2C cells) or at the tetraploid level (4C and 2C x 2 cells). The proportion of these cells was not affected by the age of the animals or the annual cycle, thus suggesting that there is no age-related increase in the mean DNA content in the frog HPR. The mean DNA contents of H2C and 4C cells were much higher than those in the standard (hepatocytes). This cannot be simply attributed to the presence of different amounts of nuclear proteins, but rather indicates that at least a certain proportion of the highest DNA contents may be due to a real extra-DNA synthesis.Dedicated to Professor Dr. T.H. Schiebler on the occasion of his 65th birthday     

Determination of DNA content in the nurse and follicle cells from wild type and mutant Drosophila melanogaster by DNA-Feulgen cytophotometry

DNA replication patterns in the nurse and follicle cells of wild type and a female sterile mutant, fs(1)1304, of Drosophila melanogaster have been studied by DNA-Feulgen cytophotometry, using a cell dispersal technique that allowed the measurement of DNA amounts in individual nuclei from egg chambers of known developmental stages. DNA-Feulgen values associated with various ovarian nuclei from egg chambers at different stages of development were used to assess a base line DNA content for ovarian tissues and to estimate the extent of DNA replication in the nurse cells and follicle cells of growing and mature egg chambers. Our data show that both the nurse and follicle cells undergo multiple cycles of endonuclear DNA replication and that there may be selective amplification as well as underreplication by portions of the genome in these highly polyploid, ovarian cells. Alternative models are proposed to account for the DNA replication patterns observed. Comparisons of DNA-Feulgen levels in wild type ovarian nuclei with those found for the fs(1)1304 mutant and its heterozygote in the balanced stock fs/FM3, show that equivalent DNA levels are present in follicle cell nuclei from all three types of females. Nurse cell nuclei in the homozygous fs stock, however, fail to achieve the same high DNA levels observed in both fs/FM3 and wild type nurse cell nuclei. Although the nuclei of follicle cells in ovaries from fs/fs females appear morphologically like those surrounding egg chambers in wild type ovaries, nurse cell nuclei from mutant females show a more compacted organization of their chromatin than found for nurse cell nuclei from wild type ovaries at similar developmental stages. Our findings suggest that a major effect of the fs(1)1304 mutation may be on the coiling behavior of chromatin and the conformation of DNA-protein moieties in both nurse cell and follicle cell nuclei. These changes in chromatin structure apparently are manifest by perturbations in DNA replication patterns and normal gene function in these biosynthetically active cells.     

Characterization of cloned ribosomal DNA from Drosophila hydei.

The structure of ribosomal genes from the fly Drosophila hydei has been analyzed. EcoRI fragments, cloned in a plasmid vector, were mapped by restriction enzyme analysis. The lengths of the regions coding for 18S and 28S rRNA were defined by R-loop formation. From these data a physical map of the rRNA genes was constructed. There are two major types of rDNA units in D. hydei, one having a size of 11 kb and the other a size of 17 kb. The 17 kb unit results from an intervening sequence (ivs) of 6.0 kb, interrupting the beta-28S rRNA coding region. Some homology between th D. hydei ivs and D. melanogaster type 1 ivs has been described previously (1). However, the restriction sites within these ivs show considerable divergence. Whereas D. hydei rDNA D. melanogaster rDNA, the nontranscribed spacer has little, if any, sequence homology. Despite difference in sequence, D. hydei and D. melanogaster spacers show structural similarities in that both contain repeated sequence elements of similar size and location.     

Biochemical,morphological and flow cytometric evaluation of the effects of hexachlorobenzene on rat liver

This study investigated the ability of HCB (0.1% in the diet for 15 days) to cause early changes in the cellular ploidy of rat liver. Treatment caused marked hepatomegaly, increase of microsomal proteins and cytochrome P-450 content and reduction of hepatocyte microviscosity. Microscopic examination showed that the hepatocytes were enlarged, with hyaline cytoplasm and vacuoles. The size distribution of the isolated hepatocytes showed a larger percentage of bigger cells. Flow-cytometric DNA/protein analysis was performed on whole (fixed) cells and on nuclei. From the combined results of both analyses it was possible to exclude significant changes in the percentages of diploid, mononucleated tetraploid, binucleated tetraploid and octoploid hepatocytes. The DNA and protein content of each subpopulation remained unchanged. Our results suggest that HCB does not cause early diploidization of liver cells and that hepatomegaly and cytochrome P-450 induction seem not to be correlated with effects on total DNA and total protein contents.Abbreviations HCB hexachlorobenzene - PI propidium iodide - FITC fluorescein isothiocyanate - DN diploid nuclei - SN 2N-4N nuclei in S-phase - TN tetraploid nuclei - DC diploid cells - SDC 2N-4N diploid cells in S-phase - TC tetraploid cells - STC 4N-8N tetraploid cells in S-phase - OC octoploid cells - MDC mononucleated diploid cells - SMDC mononucleated diploid cells in S-phase - BOC binucleated octoploid cells - SBTC binucleated tetraploid cells in S-phase - BTC binucleated tetraploid cells - MTC mononucleated tetraploid cells.     

Disproportionate numbers of rDNA loci in diploid and polyploid testis nuclei of gerris najas detected by fluorescence in situ hybridization

The replication state of rDNA in testes nuclei undergoing polyploidization by classical-type endomitosis was investigated in Gerris najas (Heteroptera) by means of fluorescence in situ hybridization. The number of just one rDNA locus per haploid genome was determined by in situ hybridization on meiotic nuclei. Additionally, DNA measurements of spermatids and testes nuclei were performed. Although regular duplication levels of nuclear DNA were found within the limits of the accuracy of the method, these did evidently not apply to the ribosomal genes. The comparison of the number of rDNA signals with the DNA content of 106 testis nuclei revealed drastic variations of the number of rDNA loci between individual nuclei with similar DNA content. Polyploid nuclei of the testis epithelium showed too low numbers of rDNA loci in relation to those expected from the levels of ploidy, while cyst cell nuclei displayed increased numbers of rDNA loci. The results indicate that the ribosomal genes are either underreplicated, or in part eliminated, during the endomitotic cycles of epithelium cell nuclei, but amplified in the cyst cell nuclei, probably already at their diploid stage.     

Intercellular cytoplasm transport during oogenesis of the moth midge, Tinearia alternata Say (Diptera: Psychodidae)

Polytrophic ovaries of the nematocerous dipteran, Tinearia alternata Say consists of several developmentally synchronized ovarioles each housing only one functional egg chamber with 15 nurse cells and an oocyte. At the early stages of previtellogenesis the nurse cells become polyploid and synthetically active. Their nuclei contain polytene chromosomes and prominent nucleoli. With the advance of previtellogenic growth the nurse cell cytoplasm is loaded with the growing number of ribosomes and contain perinuclear nuage material, mitochondria, electron dense bodies and aggregations of endoplasmic reticulum. All these organelles are transported into the oocyte thanks to the massive and rapid flow of the nurse cell cytoplasmic contents. Nurse cell-oocyte transport is mediated by actin cytoskeleton. Prior to the rapid cytoplasm transfer, F-actin network is associated with the nurse cell membranes while tiny bundles of microfilaments form actin baskets connected with ring canals. Nurse cells in Tinearia lack an extensive scaffold of radially oriented, F-actin bundles (cables) that would tether their nuclei in place, thus preventing ring canals from plugging. The way the nuclei are anchored to their central positions within the cells remains unclear. Towards the final stages of oogenesis nurse cells are almost devoid of cytoplasm and degenerate. Although their nuclei undergo dramatic morphological transformations, typical hallmarks of apoptotic pathway could not be clearly observed. Rapid ooplasmic streaming does not occur.     

半枝莲中印黄芩甙的R－HPLC分析

半枝莲中印黄芩甙的Ｒ－ＨＰＬＣ分析代雪平魏波（河南省药检所,郑州４５０００３）（河南省人民医院药剂科,郑州４５０００３）Ｒ┐ＨＰＬＣａｎａｌｙｓｉｓｏｆｓｃｕｔｅｌａｒｉｎｆｒｏｍＳｃｕｔｅｌａｒｉａｂａｒｂａｔａＤ．ＤｏｎＤａｉＸｕｅ－Ｐｉｎｇ（Ｈ...     

The intron boundaries and flanking rRNA coding sequences of Calliphora erythrocephala rDNA

We have sequenced the available cloned examples of the intron-coding sequence junctions for the rDNA of the higher Dipteran, Calliphora erythrocephala. The introns interrupt the rDNA at the same position as the type 1 intron family detected in Drosophila melanogaster and D. virilis (10,11). A duplication of 14 base pairs of the 28S rRNA coding sequence surrounds a short version of the major genomic length class of introns. This same duplication is associated with boundaries of the type 1 introns in D. virilis and D. melanogaster (10, 13,14). We have detected considerable homology between the 3' intron sequences of C. erythrocephala and D. virilis. The rRNA coding sequences flanking the introns are extremely homologous in C. erythrocephala, D. melanogaster and D. virilis, with only one small region of significant divergence. This corresponds to a variable stem region previously identified in eukaryotic 28S rRNA at a site analogous to the L1 ribosomal protein binding site of prokaryotic 23S rRNA (27).     

Reductions and new inventions dominate oogenesis of Strepsiptera (Insecta)

The endoparasitic life of strepsipterans (Insecta), especially neotenic females, reduces to a great extent external and internal organs. Light and electron microscopic investigation of ovaries of Elenchus tenuicornis (Kirby) confirms the following: (1) somatic tissues of ovaries are totally reduced, with the exception of some cells surrounding germ cell clusters; (2) a previtellogenic growth phase of oocytes is reduced; (3) nurse cells remain diploid and their membranes degenerate at the onset of vitellogenesis; (4) vitellogenesis is reduced, vitellin and fat vacuoles contribute only 50% to the final egg volume; and (5) chorionogenesis is reduced to a vitellin membrane. However, some features of normal development remain, allowing classification of the ovary type as polytrophic meroistic: (1) germ cells undergo synchronized, incomplete divisions, following the 2ⁿ rule, where all former intercellular bridges become localized in one cystocyte, while the other has none; and (2) only one cell is determined as the oocyte, all other cystocytes serve as nurse cells and the surrounding somatic cells transform into follicular cells. Novel events in oogenesis of strepsipterans include fission of clusters during the phase of cluster mitoses, and protection of oocyte nuclei, while nurse cell nuclei degenerate in the same cytoplasm.     

Underreplication of satellite DNAs in polyploid ovarian tissue of Drosophila virilis

The satellite DNAs of Drosophila virilis have been examined in diploid and polyploid tissues by isopycnic ultracentrifugation and thermal denaturation experiments. Previous work has established that the satellite DNAs are under replicated in the polytene chromosomes of the salivary glands of D. virilis. The results of the present experiments demonstrate that this underreplication also takes place in the ovaries which contain nurse cells and follicle cells. These tissues are polyploid but do not show polytene chromosomes.     

The ovaries of Mecoptera: basic similarities and one exception to the rule

Two entirely different types of ovaries (ovarioles) have been described in mecopterans. In the representatives of Meropeidae, Bittacidae, Panorpodidae and Panorpidae the ovarioles are of the polytrophic-meroistic type. Four regions: a terminal filament, germarium, vitellarium and ovariole stalk can be distinguished in the ovarioles. The germaria house numerous germ cell clusters. Each cluster arises as a result of 2 consecutive mitoses of a cystoblast and consists of 4 sibling cells. The oocyte always differentiates from one of the central cells of the cluster, whereas the remaining 3 cells develop into large, polyploid nurse cells. The vitellaria contain 7-12 growing egg chambers (= oocyte-nurse cell complexes). In contrast, the ovaries of the snow flea, Boreus hyemalis, are devoid of nurse cells and therefore panoistic (secondary panoistic). The ovarioles are composed of terminal filaments, vitellaria and ovariole stalks only; in adult females functional germaria are absent. Histochemical tests suggest that amplification of rDNA takes place in the oocyte nuclei. Resulting dense nucleolar masses undergo fragmentation into multiple polymorphic nucleoli. The classification of extant mecopterans as well as the phylogenetic relationships between Mecoptera and Siphonaptera are discussed in the context of presented data.     

Polyploid tissues in the nematode Caenorhabditis elegans

During larval development, the number of somatic nuclei in C. elegans hermaphrodites increases from 558 to 959 (J. E. Sulston and H. R. Horvitz, Dev. Biol. 56, 110-156, 1977; J. E. Sulston et al., Dev. Biol. 100, 64-119, 1983). At the same time, the animals increase about 60-fold in volume. We have measured the DNA contents of several classes of nuclei by quantitating the fluorescence of Hoescht 33258 stained DNA (D. G. Albertson et al., Dev. Biol. 63, 165-178, 1978). Probably all embryonic nuclei, including those of neurons, muscles, hypodermis, and intestine, are diploid at hatching. Neurons, muscles, and nondividing hypodermal nuclei remain diploid throughout larval development. The DNA content of the intestinal nuclei doubles at the end of each larval stage, reaching 32C by the adult stage. New hypodermal cells, generated by division of seam cells in the larval stages, undergo an additional round of DNA replication before fusing with the major syncytium (hyp7, Sulston et al., 1983). Thus the larval hyp7 syncytium comprises a fixed number of diploid embryonic nuclei plus an increasing number of tetraploid postembryonic nuclei. Some of the endoreduplications that occur in the intestinal and hypodermal lineages of C. elegans may correspond to nuclear or cellular divisions in another nematode Panagrellus redivivus (P. W. Sternberg and H. R. Horvitz, Dev. Biol. 93, 181-205, 1982).     

Determination of the region of rDNA involved in polytenization in salivary glands of Drosophila hydei

During the formation of polytene chromosomes in salivary glands of Drosophila hydei, the genes for ribosomal RNA (rDNA) are underreplicated relative to the rest of the genome. We have measured the number of rRNA genes with and without intervening sequences (ivs+ and ivs- genes) in polytene chromosomes of different genotypes. In the group of genotypes having a large number of ivs- rRNA genes polytenization only occurs within the cluster of ivs- genes. In each of these genotypes rDNA polytenization reaches a constant level of 150 ivs- genes per two chromatid sets (2C); X/X constitutions having two nucleolus organizers (NOs) in the diploid set polytenize the same amount of rDNA as X/O constitutions. In the group of genotypes with small ivs- gene numbers, the rDNA region involved in polytenization is longer and has an average length of 1,700 kb per NO, which is constant in these genotypes. Polytenization of rDNA is extended into the cluster of ivs+ genes, in spite of the fact that these genes appear to be nonfunctional. The smaller the number of ivs- genes, the greater the number of ivs+ genes that are polytenized in the NO. In these genotypes, X/X females replicate twice as much rDNA as X/O males, suggesting that both NOs of the diploid set are polytenized. A comparison of the pattern of spacer length heterogeneity in hybrids between different stocks also demonstrates that both NOs are replicated during polytenization.     

On the nurse cell and the spermatozeugma in Littorina sitkana

Summary Nurse cells develop from diploid cells in the testis. Each cell undergoes a reduction division which leaves the nucleus with half the volume of a normal diploid cell. They send out pseudopodia which form desmosomelike junctions with developing spermatids. The nurse cells detach from the testicular wall, their nuclei degenerate and secretion droplets form in the cytoplasm. The pseudopodia are drawn in as the cytoplasmic secretions swell and the nurse cell becomes spherical. The eupyrene sperm become grouped unilaterally and at this stage are attached to the nurse cell by only the tips of their acrosomes. At maturity the nurse cells with their clumps of attached eupyrene sperm (spermatozeugmata) are released from the testis via ducts into the seminal vesicles, where they are stored prior to copulation. Nurse cells serve similar functions to those of apyrene sperm which are common among the Molluscs. We believe that the nurse cell and apyrene sperm are homologous.