Ultrastructural localization of 5-methylcytosine on DNA and RNA

DNA methylation is the major epigenetic modification and it is involved in the negative regulation of gene expression. Its alteration can lead to neoplastic transformation. Several biomolecular approaches are nowadays used to study this modification on DNA, but also on RNA molecules, which are known to play a role in different biological processes. RNA methylation is one of the most common RNA modifications and 5-methylcytosine presence has recently been suggested in mRNA. However, an analysis of nucleic acid methylation at electron microscope is still lacking. Therefore, we visualized DNA methylation status and RNA methylation sites in the interphase nucleus of HeLa cells and rat hepatocytes by ultrastructural immunocytochemistry and cytochemical staining. This approach represents an efficient alternative to study nucleic acid methylation. In particular, this ultrastructural method makes the visualization of this epigenetic modification on a single RNA molecule possible, thus overcoming the technical limitations for a (pre-)mRNA methylation analysis.     

Follicles development in the foetal human ovary.

In the foetal human ovary, diameters of oocyte and follicle, as well as those of oocyte and nucleus, are found to be positively and linearly correlated with each other. Follicle diameter and number of granulosa cells also show a positive and linear relationship. Finally, in all ovaries examined, from 5 months after conception onwards, small antral follicles were assessed.     

Follicles development in the foetal human ovary

Summary In the foetal human ovary, diameters of oocyte and follicle, as well as those of oocyte and nucleus, are found to be positively and linearly correlated with each other. Follicle diameter and number of granulosa cells also show a positive and linear relationship. Finally, in all ovaries examined, from 5 months after conception onwards, small antral follicles were assessed.     

Friedrich Miescher Prize awardee lecture review. A conserved family of nuclear export receptors mediates the exit of messenger RNA to the cytoplasm

The distinguishing feature of eukaryotic cells is the segregation of RNA biogenesis and DNA replication in the nucleus, separate from the cytoplasmic machinery for protein synthesis. As a consequence, messenger RNAs (mRNAs) and all cytoplasmic RNAs from nuclear origin need to be transported from their site of synthesis in the nucleus to their final cytoplasmic destination. Nuclear export occurs through nuclear pore complexes (NPCs) and is mediated by saturable transport receptors, which shuttle between the nucleus and cytoplasm. The past years have seen great progress in the characterization of the mRNA export pathway and the identification of proteins involved in this process. A novel family of nuclear export receptors (the NXF family), distinct from the well-characterized family of importin β-like proteins, has been implicated in the export of mRNA to the cytoplasm. Received 23 January 2001; received after revision 12 April 2001; accepted 12 April 2001     

Organization of the oocyte nucleus in silphid beetles (Silphidae,Coleoptera-Polyphaga)

Summary In all the Silphid species examined, the oocyte chromosomes were found to, contract to form the karyospheres. Despite this, as the oocytes grow the germinal vesicles increase also very considerably. Production of numerous RNA-containing nucleolus-like bodies was found to be characteristic of the Silphid oogenesis. Experiments with³H-uridine demonstrated that only the karyosphere actively synthesized RNA, while the nucleolus-like bodies remained inactive in this process.This investigation was supported in part under Contract DPKBN/52/76-II. 1.3.10. with the Polish Academy of Science.     

Biochemical alterations in the oocyte in support of early embryonic development

Notwithstanding the enormous reproductive potential encapsulated within a mature mammalian oocyte, these cells present only a limited window for fertilization before defaulting to an apoptotic cascade known as post-ovulatory oocyte aging. The only cell with the capacity to rescue this potential is the fertilizing spermatozoon. Indeed, the union of these cells sets in train a remarkable series of events that endows the oocyte with the capacity to divide and differentiate into the trillions of cells that comprise a new individual. Traditional paradigms hold that, beyond the initial stimulation of fluctuating calcium (Ca²⁺) required for oocyte activation, the fertilizing spermatozoon plays limited additional roles in the early embryo. While this model has now been drawn into question in view of the recent discovery that spermatozoa deliver developmentally important classes of small noncoding RNAs and other epigenetic modulators to oocytes during fertilization, it is nevertheless apparent that the primary responsibility for oocyte activation rests with a modest store of maternally derived proteins and mRNA accumulated during oogenesis. It is, therefore, not surprising that widespread post-translational modifications, in particular phosphorylation, hold a central role in endowing these proteins with sufficient functional diversity to initiate embryonic development. Indeed, proteins targeted for such modifications have been linked to oocyte activation, recruitment of maternal mRNAs, DNA repair and resumption of the cell cycle. This review, therefore, seeks to explore the intimate relationship between Ca²⁺ release and the suite of molecular modifications that sweep through the oocyte to ensure the successful union of the parental germlines and ensure embryogenic fidelity.     

Regulatory mechanisms of RNA function: emerging roles of DNA repair enzymes

The acquisition of an appropriate set of chemical modifications is required in order to establish correct structure of RNA molecules, and essential for their function. Modification of RNA bases affects RNA maturation, RNA processing, RNA quality control, and protein translation. Some RNA modifications are directly involved in the regulation of these processes. RNA epigenetics is emerging as a mechanism to achieve dynamic regulation of RNA function. Other modifications may prevent or be a signal for degradation. All types of RNA species are subject to processing or degradation, and numerous cellular mechanisms are involved. Unexpectedly, several studies during the last decade have established a connection between DNA and RNA surveillance mechanisms in eukaryotes. Several proteins that respond to DNA damage, either to process or to signal the presence of damaged DNA, have been shown to participate in RNA quality control, turnover or processing. Some enzymes that repair DNA damage may also process modified RNA substrates. In this review, we give an overview of the DNA repair proteins that function in RNA metabolism. We also discuss the roles of two base excision repair enzymes, SMUG1 and APE1, in RNA quality control.     

Gold nanoparticles induce nuclear damage in breast cancer cells,which is further amplified by hyperthermia

Gold nanoparticles have emerged as promising tools for cancer research and therapy, where they can promote thermal killing. The molecular mechanisms underlying these events are not fully understood. The geometry and size of gold nanoparticles can determine the severity of cellular damage. Therefore, small and big gold nanospheres as well as gold nanoflowers were evaluated side-by-side. To obtain quantitative data at the subcellular and molecular level, we assessed how gold nanoparticles, either alone or in combination with mild hyperthermia, altered the physiology of cultured human breast cancer cells. Our analyses focused on the nucleus, because this organelle is essential for cell survival. We showed that all the examined gold nanoparticles associated with nuclei. However, their biological effects were quantitatively different. Thus, depending on the shape and size, gold nanoparticles changed multiple nuclear parameters. They redistributed stress-sensitive regulators of nuclear biology, altered the nuclear morphology, reorganized nuclear laminae and envelopes, and inhibited nucleolar functions. In particular, gold nanoparticles reduced the de novo biosynthesis of RNA in nucleoli, the subnuclear compartments that produce ribosomes. While small gold nanospheres and nanoflowers, but not big gold nanospheres, damaged the nucleus at normal growth temperature, several of these defects were further exacerbated by mild hyperthermia. Taken together, the toxicity of gold nanoparticles correlated with changes in nuclear organization and function. These results emphasize that the cell nucleus is a prominent target for gold nanoparticles of different morphologies. Moreover, we demonstrated that RNA synthesis in nucleoli provides quantitative information on nuclear damage and cancer cell survival.     

The expression of Sam68, a protein involved in insulin signal transduction,is enhanced by insulin stimulation

The role of Sam68, an RNA binding protein and putative substrate of the insulin receptor (IR) in insulin signaling was studied using CHO wild type (WT) cells, CHO cells overexpressing IR, and rat white adipocytes as a physiological system. In CHO-IR cells and adipocytes, Sam68 was tyrosine phosphorylated in response to insulin, and then associated with p85 phosphatidylinositol-3 kinase along with IRS-1. Sam68 was localized mainly in the nucleus of CHO-WT, and both in the nucleus and cytoplasm of CHO-IR cells, but only in the cytoplasm of rat white adipocytes. Insulin stimulation for 16 h enhanced the expression of Sam68 in rat adipocytes and CHO-IR cells. Moreover, CHO-IR cells expressed more Sam68 than CHO-WT, suggesting that overexpression of the IR is enough to induce the expression of Sam68. In summary, these results demonstrate that Sam68 works as a cytoplasmic docking protein which is recruited by IR signaling and whose expression is induced by insulin stimulation, suggesting a putative role for Sam68 in insulin signal transduction.     

3H-thymidine incorporation (DNA synthesis) and radiotoxicity in the ovary of the Japanese quail before and during follicle formation

No evidence was found for ribosomal DNA amplification in the oocytes of the Japanese quail, before or during folliculogenesis. DNA synthesis in the somatic cells, involved in follicle formation, starts at the medullar side of the basement membrane. The localized sterilization of the quail ovary after administration of 3H-thymidine (3H-TdR) seems to be due to radiation-induced lesions in the follicle forming somatic cells, rather than to direct radiation damage of the oocyte.     

Transgenesis in fish

Gene transfer into fish embryo is being performed in several species (trout, salmon, carps, tilapia, medaka, goldfish, zebrafish, loach, catfish, etc.). In most cases, pronuclei are not visible and microinjection must be done into the cytoplasm of early embryos. Several million copies of the gene are generally injected. In medaka, transgenesis was attempted by injection of the foreign gene into the nucleus of oocyte. Several reports indicate that the injected DNA was rapidly replicated in the early phase of embryo development, regardless of the origin and the sequence of the foreign DNA. The survival of the injected embryos was reasonably good and a large number reached maturity. The proportion of transgenic animals ranged from 1 to 50% or more, according to species and to experimentators. The reasons for this discrepancy have not been elucidated. In all species, the transgenic animals were mosaic. The copy number of the foreign DNA was different in the various tissues of an animal and a proportion lower than 50% of F1 offsprings received the gene from their parents. This suggests that the foreign DNA was integrated into the fish genome at the two cells stage or later. An examination of the integrated DNA in different cell types of an animal revealed that integration occurred mainly during early development. The transgene was found essentially unrearranged in the fish genome of the founders and offsprings. The transgenes were therefore stably transmitted to progeny in a Mendelian fashion. Southern blot analysis revealed the presence of possible junction fragments and also of minor bands which may result from a rearrangement of the injected DNA. In all species, the integrated DNA appeared mainly as random end-to-end concatemers. In adult trout blood cells, a small proportion of the foreign DNA was maintained in the form of non-integrated concatemers, as judged by the existence of end fragments. The transgenes were generally only poorly expressed. The majority of the injected gene constructs contained essentially mammalian or higher vertebrates sequences. The comparison of the expression efficiency of these constructs in transfected fish and mammalian cells indicates that some of the mammalian DNA sequences are most efficiently understood by the fish cell machinery. Chloramphenicol acetyl transferase gene under the control of promoters from Rous sarcoma virus, and human cytomegalovirus, was expressed in several tissues of transgenic fish. Chicken delta-crystallin gene was expressed in several tissues of transgenic fish.(ABSTRACT TRUNCATED AT 400 WORDS)     

Vitellogenesis in the air breathing fish,Channa punctatus (Bloch)

Summary Two types of yolk develop in the oocyte ofChanna punctatus. The carbohydrate yolk, which develops from the material present in the ooplasm, breaks up for the use of the growing oocyte before ovulation takes place. The proteid yolk, developing from the extraoocytic material, finally crams the fully mature oocyte, perhaps to participate in the process of embryogenesis.Acknowledgments. The authors are grateful to Professor C.C. Das, for his encouragement and the laboratory facilities. The award of a U.G.C. junior fellowship to one of us (U.R.A.) by the Berhampur University is thankfully acknowledged.     

Vergleichende Elektronenmikroskopie reiner DNS und der DNS des Bakteriennukleoids

Summary The behaviour of pure isolated DNA towards the conditions of fixation and dehydration—prior to ultrathin sectioning—has been studied and compared with the behaviour of the DNA-plasm of the bacterial nucleus. It is found that those conditions which produce coarse coagulation of the free DNA also produce electron opaque bodies of variable aspect inside the bacterial nucleus. The coagulation figures obtained in both situations are very similar. This is a further direct evidence in favour of the view that the bacterial nucleus is an assembly of fine DNA-containing fibrilla in a highly hydrated plasma.     

RNA fragments rich in G and A nucleotides obligatory for in vitro DNA replication of phages phi-X 174 and lambda]

Evidence was presented that in vitro conversion of single-stranded DNA of phage phi X 174 to the double-stranded replicative form by partially purified DNA-dependent DNA polymerase I requires a specific RNA fragment acting as primer (25-50 nucleotides). RNA fragments highly rich in nucleotides A and G were obtained by partial degradation of E. coli M 500 Sho-R ribosomal RNA with pancreatic ribonuclease. They become covalently bound to the newly synthesized DNA chain of the replicative form of phage phi X 174. These RNA fragments are also required for in vitro replication of lambda phage DNA.     

The capacity of oocytes for DNA repair

Female fertility and offspring health are critically dependent on the maintenance of an adequate supply of high-quality oocytes. Like somatic cells, oocytes are subject to a variety of different types of DNA damage arising from endogenous cellular processes and exposure to exogenous genotoxic stressors. While the repair of intentionally induced DNA double strand breaks in gametes during meiotic recombination is well characterised, less is known about the ability of oocytes to repair pathological DNA damage and the relative contribution of DNA repair to oocyte quality is not well defined. This review will discuss emerging data suggesting that oocytes are in fact capable of efficient DNA repair and that DNA repair may be an important mechanism for ensuring female fertility, as well as the transmission of high-quality genetic material to subsequent generations.     

L'effet de la basse température sur l'incorporation de précurseurs des acides nucléiques et des protéines dans les cellules du méristème radiculaire deVicia faba

Summary The uptake of labelled RNA and protein precursors inVicia faba root meristems into nuclei at 4°C is less depressed than in the cytoplasm. Reversion of the normal pattern of incorporation would seem to indicate an inhibition of the RNA transfer from nucleus to cytoplasm.