Restoration of pole-cell-forming ability to u.v.-irradiated Drosophila embryos by injection of mitochondrial lrRNA

Development ◽  
1989 ◽  
Vol 107 (4) ◽  
pp. 733-742 ◽  
Author(s):  
S. Kobayashi ◽  
M. Okada
Keyword(s):  
Dna Sequences ◽  
Nuclear Dna ◽  
Mitochondrial Gene ◽  
Cell Formation ◽  
Nuclear Bodies ◽  
Cdna Insert ◽  
Pole Cell ◽  
Polar Granules ◽  
Pole Cells

Screening a cDNA library generated from poly(A) +RNA of Drosophila cleavage embryos, we selected a cDNA clone (pDE20.6). The cDNA hybridized specifically with a poly(A) +RNA that is capable of restoring embryos from u.v.-caused inability of pole cell formation. The RNA hybrid-selected by pDE20.6 was also able to induce pole cells in the anterior region of embryos, if it was coinjected with u.v.-irradiated polar plasm, although the RNA or irradiated polar plasm alone was not effective. Pole cells thus formed in the anterior or in the u.v.-irradiated posterior region were identified by polar granules and nuclear bodies, morphological markers for normal pole cells. Furthermore, the RNA-induced pole cells were able to migrate into gonadal rudiments. The nucleotide sequence of pDE20.6 cDNA insert was highly homologous with the mitochondrial large rRNA (lrRNA) gene, but not with any nuclear DNA sequences. Using pDE20.6 as a primer, a full-length cDNA of mitochondrial lrRNA was generated and cloned. The RNA transcribed in vitro from the cDNA was able to restore pole cell formation. The cDNA hybridized only with a 1.5 kb poly(A) +RNA on a Northern blot. The 1.5 kb RNA sedimented more with the post-mitochondrial (P3) fraction than with the mitochondrial (P2) fraction, while the majority of transcripts from another mitochondrial gene was detected in the P2 fraction.

scholarly journals Identification of a component of Drosophila polar granules

Development ◽  
1988 ◽  
Vol 103 (4) ◽  
pp. 625-640 ◽  
Author(s):  
B. Hay ◽  
L. Ackerman ◽  
S. Barbel ◽  
L.Y. Jan ◽  
Y.N. Jan
Keyword(s):  
Germ Line ◽  
Posterior Pole ◽  
Nuclear Bodies ◽  
Maternal Origin ◽  
Pole Cell ◽  
Polar Granules ◽  
Early Embryos ◽  
Biochemical Studies ◽  
Males And Females ◽  
Pole Cells

Information necessary for the formation of pole cells, precursors of the germ line, is provided maternally and localized to the posterior pole of the Drosophila egg. The maternal origin and posterior localization of polar granules suggest that they may be associated with pole cell determinants. We have generated an antibody (Mab46F11) against polar granules. In oocytes and early embryos, the Mab46F11 antigen is sharply localized to the posterior embryonic pole. In pole cells, it becomes associated with nuclear bodies within, and nuage around, the nucleus. Immunoreactivity remains associated with cells of the germ line throughout the life cycle of both males and females. This antibody recognizes a 72–74 × 10(3) Mr protein and is useful both as a pole lineage marker and in biochemical studies of polar granules.

scholarly journals Transcriptomic and functional analysis of the oosome, a unique form of germ plasm in the waspNasonia vitripennis

2018 ◽  
Author(s):  
Honghu Quan ◽  
Jeremy Lynch
Keyword(s):  
Molecular Basis ◽  
Germ Plasm ◽  
Cell Formation ◽  
Solid Sphere ◽  
The Novel ◽  
Developmental Networks ◽  
Pole Cell ◽  
Polar Granules ◽  
Developmental Role ◽  
Pole Cells

AbstractBackgroundThe oosome is the germline determinant in the waspNasonia vitripennisand is homologous to the polar granules ofDrosophila. Despite a common evolutionary origin and developmental role, the oosome is morphologically quite distinct from polar granules. It is a solid sphere that migrates within the cytoplasm before budding out and forming pole cells.ResultsTo gain an understanding of both the molecular basis of the novel form of the oosome, and the conserved essential features of germ plasm, we quantified and compared transcript levels between embryo fragments that contained the oosome, and those that did not. The identity of the localized transcripts indicated thatNasoniauses different molecules to carry out conserved germ plasm functions. In addition, functional testing of a sample of localized transcripts revealed potentially novel mechanisms of ribonucleoprotein assembly and pole cell cellularization in the wasp.ConclusionsOur results demonstrate that numerous novel and unexpected molecules have been recruited in order produce the unique characteristics of the oosome and pole cell formation inNasonia. This work will serve as the basis for further investigation into the patterns of germline determinant evolution among insects, the molecular basis of extreme morphology of ribonucleoproteins, and the incorporation of novel components into developmental networks.

Polar granules and pole cells in the embryo of Calliphora erythrocephala: ultrastructure and [3H]leucine labelling

Development ◽  
1980 ◽  
Vol 57 (1) ◽  
pp. 79-93
Author(s):  
Anders Lundquist ◽  
Hadar Emanuelsson
Keyword(s):  
Cell Formation ◽  
Electron Microscopic ◽  
Electron Microscopic Autoradiography ◽  
Calliphora Erythrocephala ◽  
Pole Cell ◽  
Polar Granules ◽  
Pole Cells ◽  
Autoradiographic Analysis ◽  
Pole Plasm ◽  
Blastoderm Stage

The polar granules in Calliphora undergo a gradual fragmentation during early cleavage, but reaggregate after pole-cell formation. Autoradiographic analysis showed that the pole cells in Calliphora acquire a higher ‘3H’leucine label than the rest of the embryo during the blastoderm stage. Such an increased label was not seen in the pole plasm before pole-cell formation or in the pole cells during gastrulation. Electron microscopic autoradiography revealed that the polar granules are substantially labelled during the blastoderm stage. At the same time, characteristic nuclear blebs appear in the pole cells. The observations are consistent with the hypothesis that polar granules contain maternalmessenger RNA, which is released and translated into proteins.

Transcriptional and posttranscriptional regulation of maize mitochondrial gene expression

1991 ◽  
Vol 11 (1) ◽  
pp. 533-543
Author(s):  
R M Mulligan ◽  
P Leon ◽  
V Walbot
Keyword(s):  
Dna Sequences ◽  
Transcription Initiation ◽  
Posttranscriptional Regulation ◽  
Rank Order ◽  
Mitochondrial Gene ◽  
Initiation Site ◽  
Gene Copy ◽  
Promoter Strength ◽  
Protein Coding

Lysed maize mitochondria synthesize RNA in the presence of radioactive nucleoside triphosphates, and this assay was utilized to compare the rates of transcription of seven genes. The rates of incorporation varied over a 14-fold range, with the following rank order: 18S rRNA greater than 26S rRNA greater than atp1 greater than atp6 greater than atp9 greater than cob greater than cox3. The products of run-on transcription hybridized specifically to known transcribed regions and selectively to the antisense DNA strand; thus, the isolated run-on transcription system appears to be an accurate representation of endogenous transcription. Although there were small differences in gene copy abundance, these differences cannot account for the differences in apparent transcription rates; we conclude that promoter strength is the main determinant. Among the protein coding genes, incorporation was greatest for atp1. The most active transcription initiation site of this gene was characterized by hybridization with in vitro-capped RNA and by primer extension analyses. The DNA sequences at this and other transcription initiation sites that we have previously mapped were analyzed with respect to the apparent promoter strengths. We propose that two short sequence elements just upstream of initiation sites form at least a portion of the sequence requirements for a maize mitochondrial promoter. In addition to modulation at the level of transcription, steady-state abundance of protein-coding mRNAs varied over a 20-fold range and did not correlate with transcriptional activity. These observations suggest that posttranscriptional processes are important in the modulation of mRNA abundance.

Spatial and developmental changes in the respiratory activity of mitochondria in early Drosophila embryos

Development ◽  
1992 ◽  
Vol 115 (4) ◽  
pp. 1175-1182 ◽  
Author(s):  
T. Akiyama ◽  
M. Okada
Keyword(s):  
Respiratory Activity ◽  
Cell Formation ◽  
Rhodamine 123 ◽  
Mitochondrial Activity ◽  
Early Cleavage ◽  
Pole Cell ◽  
Posterior Group ◽  
Cleavage Stages ◽  
Pole Cells ◽  
Drosophila Embryos

Mitochondria of early Drosophila embryos were observed with a transmission electron microscope and a fluorescent microscope after vital staining with rhodamine 123, which accumulates only in active mitochondria. Rhodamine 123 accumulated particularly in the posterior pole region in early cleavage embryos, whereas the spatial distribution of mitochondria in an embryo was uniform throughout cleavage stages. In late cleavage stages, the dye showed very weak and uniform accumulation in all regions of periplasm. Polar plasm, sequestered in pole cells, restored the ability to accumulate the dye. Therefore, it is concluded that the respiratory activity of mitochondria is higher in the polar plasm than in the other regions of periplasm in early embryos, and this changes during development. The temporal changes in rhodamine 123-staining of polar plasm were not affected by u.v. irradiation at the posterior of early cleavage embryos at a sufficient dosage to prevent pole cell formation. This suggests that the inhibition of pole cell formation by u.v. irradiation is not due to the inactivation of the respiratory activities of mitochondria. In addition, we found that the anterior of Bicaudal-D mutant embryos at cleavage stage was stained with rhodamine 123 with the same intensity as the posterior of wild-type embryos. No pole cells form in the anterior of Bic-D embryos, where no restoration of mitochondrial activity occurs in the blastoderm stage. The posterior group mutations that we tested (staufen, oskar, tudor, nanos) and the terminal mutation (torso) did not alter staining pattern of the posterior with rhodamine 123.

Differentiation and positioning of nuclei in eggs of the cecidomyid Heteropeza pygmaea

1976 ◽  
Vol 22 (1) ◽  
pp. 99-113
Author(s):  
M. Meats ◽  
J.B. Tucker
Keyword(s):  
Structural Changes ◽  
Early Stage ◽  
Cell Formation ◽  
Longitudinal Axis ◽  
Posterior Pole ◽  
Spindle Orientation ◽  
Specific Sequence ◽  
Pole Cell ◽  
Polar Granules ◽  
Egg Chamber

During the first three cleavage divisions of the egg nuclei a precise sequence of spindle orientation and elongation parallel to the longitudinal axis of the egg is apparently involved in positioning one nucleus among the polar granules at the posterior pole of the egg. The size of this nucleus, and the position at which the egg cleaves when pole cell formation occurs, appear to constitute part of the mechanism which ensures that only one nucleus is included in the first pole cell. Blastoderm formation occurs without a well-defined migration of nuclei to the egg surface. Nuclei are so large in relation to the size of the egg that uniform spacing and distribution of nuclei ensures that a large proportion are situated near the egg surface. Those nuclei which are near the egg surface divide synchronously to form a layer of blastoderm nuclei, while membranous cleavage furrows invaginate from the egg surface between them. Nuclei in the central region of the egg chamber condense to form yolk nuclei before blastoderm nuclei have been separated from the rest of the egg by the completion of the cleavage membranes. Polar granules provide the only evidence of fine-structural differences in different regions of the egg chamber cytoplasm. They are found near the posterior pole of the egg from an early stage of oogenesis. They undergo a specific sequence of structural changes and increase in size as the egg grows. No microtubular or microfibrillar arrays have been found in the egg chamber which might form a cytoskeletal basis for spindle orientation or for the spatial differences which develop during differentiation of the uncleaved egg cytoplasm.

Use of yeast nuclear DNA sequences to define the mitochondrial RNA polymerase promoter in vitro

1989 ◽  
Vol 9 (8) ◽  
pp. 3193-3202
Author(s):  
G T Marczynski ◽  
P W Schultz ◽  
J A Jaehning
Keyword(s):  
Rna Polymerase ◽  
Dna Sequences ◽  
Nuclear Dna ◽  
In Vitro Transcription ◽  
Catalytic Rna ◽  
Mitochondrial Rna ◽  
Promoter Sequences ◽  
Sequence Recognition ◽  
Mitochondrial Rna Polymerase

We have extended an earlier observation that the TATA box for the nuclear GAL10 gene serves as a promoter for the mitochondrial RNA polymerase in in vitro transcription reactions (C. S. Winkley, M. J. Keller, and J. A. Jaehning, J. Biol. Chem. 260:14214-14223, 1985). In this work, we demonstrate that other nuclear genes also have upstream sequences that function in vitro as mitochondrial RNA polymerase promoters. These genes include the GAL7 and MEL1 genes, which are regulated in concert with the GAL10 gene, the sigma repetitive element, and the 2 microns plasmid origin of replication. We used in vitro transcription reactions to test a large number of nuclear DNA sequences that contain critical mitochondrial promoter sequences as defined by Biswas et al. (T. K. Biswas, J. C. Edwards, M. Rabinowitz, and G. S. Getz, J. Biol. Chem. 262:13690-13696, 1987). The results of these experiments allowed us to extend the definition of essential promoter elements. This extended sequence, -ACTATAAACGatcATAG-, was frequently found in the upstream regulatory regions of nuclear genes. On the basis of these observations, we hypothesized that either (i) a catalytic RNA polymerase related to the mitochondrial enzyme functions in the nucleus of the yeast cell or (ii) a DNA sequence recognition factor is shared by the two genetic compartments. By using cells deficient in the catalytic core of the mitochondrial RNA polymerase (rpo41-) and sensitive assays for transcripts initiating from the nuclear promoter sequences, we have conclusively ruled out a role for the catalytic RNA polymerase in synthesizing transcripts from all of the nuclear sequences analyzed. The possibility that a DNA sequence recognition factor functions in both the nucleus and the mitochondria remains to be tested.

scholarly journals Microhomology-mediated end joining is the principal mediator of double-strand break repair during mitochondrial DNA lesions

2016 ◽  
Vol 27 (2) ◽  
pp. 223-235 ◽  
Author(s):  
Satish Kumar Tadi ◽  
Robin Sebastian ◽  
Sumedha Dahal ◽  
Ravi K. Babu ◽  
Bibha Choudhary ◽  
...  
Keyword(s):  
Mitochondrial Dna ◽  
Dna Sequences ◽  
Nuclear Dna ◽  
Ex Vivo ◽  
Strand Break ◽  
Mitochondrial Disorders ◽  
Dna Lesions ◽  
End Joining ◽  
Dsb Repair

Mitochondrial DNA (mtDNA) deletions are associated with various mitochondrial disorders. The deletions identified in humans are flanked by short, directly repeated mitochondrial DNA sequences; however, the mechanism of such DNA rearrangements has yet to be elucidated. In contrast to nuclear DNA (nDNA), mtDNA is more exposed to oxidative damage, which may result in double-strand breaks (DSBs). Although DSB repair in nDNA is well studied, repair mechanisms in mitochondria are not characterized. In the present study, we investigate the mechanisms of DSB repair in mitochondria using in vitro and ex vivo assays. Whereas classical NHEJ (C-NHEJ) is undetectable, microhomology-mediated alternative NHEJ efficiently repairs DSBs in mitochondria. Of interest, robust microhomology-mediated end joining (MMEJ) was observed with DNA substrates bearing 5-, 8-, 10-, 13-, 16-, 19-, and 22-nt microhomology. Furthermore, MMEJ efficiency was enhanced with an increase in the length of homology. Western blotting, immunoprecipitation, and protein inhibition assays suggest the involvement of CtIP, FEN1, MRE11, and PARP1 in mitochondrial MMEJ. Knockdown studies, in conjunction with other experiments, demonstrated that DNA ligase III, but not ligase IV or ligase I, is primarily responsible for the final sealing of DSBs during mitochondrial MMEJ. These observations highlight the central role of MMEJ in maintenance of mammalian mitochondrial genome integrity and is likely relevant for deletions observed in many human mitochondrial disorders.

scholarly journals Three distinct distributions of F-actin occur during the divisions of polar surface caps to produce pole cells in Drosophila embryos.

1985 ◽  
Vol 100 (4) ◽  
pp. 1010-1015 ◽  
Author(s):  
R M Warn ◽  
L Smith ◽  
A Warn
Keyword(s):  
Cell Formation ◽  
Contractile Ring ◽  
Polar Surface ◽  
Ring Type ◽  
Actin Organization ◽  
Polar Caps ◽  
Pole Cell ◽  
Nuclear Cycle ◽  
Pole Cells ◽  
Drosophila Embryos

The F-actin distribution was studied during pole cell formation in Drosophila embryos using the phalloidin derivative rhodaminyl-lysine-phallotoxin. Nuclei were also stained with 4'-6 diamidine-2-phenylindole dihydrochloride to correlate the pattern seen with the nuclear cycle. The precursors of the pole cells, the polar surface caps, were found to have an F-actin-rich cortex distinct from that of the rest of the embryo surface and an interior cytoplasm that was less intensely stained but brighter than the cytoplasm deeper in the embryo. They were found to divide once without forming true cells and then a second time when cells formed as a result of a meridional and a basal cleavage. Three distinct distributions of the cortical F-actin have been identified during these cleavages. It is concluded that the first division, which cleaves the polar caps but does not separate them from the embryo, involves very different processes from those that lead to the formation of the pole cells. A contractile-ring type of F-actin organization may not be present during the first cleavage but is suggested to occur during the second.

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