Translesion DNA synthesis-driven mutagenesis in very early embryogenesis of fast cleaving embryos

In early embryogenesis of fast cleaving embryos, DNA synthesis is short and surveillance mechanisms preserving genome integrity are inefficient, implying the possible generation of mutations. We have analyzed mutagenesis in Xenopus laevis and Drosophila melanogaster early embryos. We report the occurrence of a high mutation rate in Xenopus and show that it is dependent upon the translesion DNA synthesis (TLS) master regulator Rad18. Unexpectedly, we observed a homology-directed repair contribution of Rad18 in reducing the mutation load. Genetic invalidation of TLS in the pre-blastoderm Drosophila embryo resulted in reduction of both the hatching rate and single-nucleotide variations on pericentromeric heterochromatin in adult flies. Altogether, these findings indicate that during very early Xenopus and Drosophila embryos TLS strongly contributes to the high mutation rate. This may constitute a previously unforeseen source of genetic diversity contributing to the polymorphisms of each individual with implications for genome evolution and species adaptation.     

The in vitro conversion of a specific molecular form of glucose 6-phosphate dehydrogenase from Musca domestica L.

A nicotinamide adenine dinucleotide phosphate dependent glucose 6-phosphate dehydrogenase (G6PD), belonging to type I of Kamada and Hori's classification, is present on the zymograms of newly emerged males of Musca domestica. It is capable of undergoing tryptic degradation and being thus transformed into a different active enzymatic form, with some of its catalytic properties unchanged, but with different electrophoretic mobility. We show in this paper that this specific G6PD form of gut origin in M. domestica is not a tissue-specific enzyme, but rather a product of hydrolytic degradation by gut proteinases which act during the process of homogenization. Besides, the G6PD of type I in the housefly is shown to be sensitive to the "storage effect" and to protection by mercaptoethanol, contrary to its hydrolytic gut form which is not sensitive to these processes. In this connection, we discuss the possible reasons for these differences in behavior.     

Mapping fungal ion channel locations

Ion channel mapping techniques are described and the results for two fungal organisms, Saprolegnia ferax and Neurospora crassa, are presented. In these species, two channel types have been characterized, stretch-activated channels exhibiting significant calcium permeability and spontaneous channels having significant potassium permeability. Two distinct analyses of patch clamp data, analysis of channel self-clustering and association between different channel types, and localization along the hyphae, reveal significant differences between the two organisms. S. ferax maintains a tip-high gradient of both channel types which is lost after disruption of the actin cytoskeleton. There is significant self-clustering of the channels, as well as interactions between channel types. N. crassa on the other hand does not maintain tip-high gradients, and clustered distributions are observed only for the stretch-activated channels. In terms of physiological roles, evidence is quite strong that the stretch-activated channels function as a growth sensor in S. ferax, but have an unknown function in N. crassa. In both organisms, the potassium permeable channels presumably function in potassium uptake. The differences between these two organisms may be due, in part, to differences in their normal environment: aquatic versus terrestrial. Copyright 1998 Academic Press.     

Map position of Aldox (Aldehyde-oxidase) and Adh (Alcohol dehydrogenase) loci on autosome II of Musca domestica L.

The Aldox and Adh structural loci of Musca domestica L. belong to autosome II. They code for the enzymes aldehyde oxidase and alcohol dehydrogenase. Both these enzymes have allelic variants with specific electrophoretic mobility, which, on cellogel, are seen as single bands. The Aldox and Adh loci encompass a large map interval, which includes the morphological markers ar, cm, and car. The recombination frequencies between these five loci indicate the alignment Aldox-ar-cm-car-Adh.

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Localisation chez Musca domestica L. des gènes Aldox et Adh codant les enzymes ald\;ehyde oxydase (AO) et alcool déshydrogénase (ADH)

Résumé Chez la mouche, les enzymes AO et ADH, observées par électrophorèse sur cellogel, présentent toutes 2 des formes alléliques exprimées par des bandes ayant une mobilité anodique propre.Les gènes structuraux Aldox et Adh, codant ces formes, sont liés entre eux et situés sur le chromosome 2. Ils se recombinent avec une fréquence élevée d'interchange; ils sont donc séparés par un intervalle important dans lequel sont compris les caractères visibles ar, cm, car. La fréquence des recombinaisons entre caractères visibles et gènes enzymatiques indique l'ordre suivant sur ce segment du deuxième chromosome de la mouche: Aldox, ar, cm, car, Adh.

     

First evidence of cell division in circulating haemocytes from the Manila clam Tapes philippinarum

In the present study, we report on haemocyte distribution, determined by a Coulter Counter, in the clam Tapes philippinarum. In addition, cytoskeleton components of haemocytes were examined using specific probes for F-actin and alpha-tubulin. The mean number of circulating haemocytes was 5 (x10(6))cells/ml haemolymph. Two main haemocyte populations were found in the haemolymph: small cells, 2-3microm in diameter and 10-100fl in volume; and large cells, 6-10microm in diameter and 150-400fl in volume. Analysis of the haemocyte cytoskeleton revealed bundles of actin filaments oriented according to the cell major axis, and microtubules radiating from the microtubule-organizing centre in proximity of the nucleus. Interestingly, mitotic spindles were also found radiating from the microtubule-organizing centres, located at the spindle poles (centrosomes) of undifferentiated cells. On the basis of both our previous findings regarding circulating stem cells (Cima, F., Matozzo, V., Marin, M.G., Ballarin, L., 2000. Haemocytes of the clam Tapes philippinarum (Adams & Reeve, 1850): morphofunctional characterisation. Fish Shellfish Immunol 10, 677-693) and new information from the present study, we suggest that haemoblasts are able to divide in the haemolymph of T. philippinarum. To our knowledge, this is the first report of mitotic spindles in circulating haemocytes from a bivalve species.     

Gut ultrastructure of the appendicularian Oikopleura dioica (Tunicata)

Abstract. The appendicularians, planktonic tunicates, possess a specialized, external filtering system that captures food particles <1 μm in size. In this work the alimentary canal of Oikopleura dioica has been studied by serial sections of whole animals and ultrastructure. The gut includes a dorsal esophagus, a bilobed saccular stomach, and a curved intestine, divided into vertical, mid-, and distal intestine (or rectum). No multicellular glands or cellular proliferative centers were found. Three main cell types were recognized, ciliated microvillar cells, globular cells and gastric band cells, with specializations reflecting different physiological roles in the various regions. Ciliated microvillar cells, the most diffuse, are considered to be involved in food propulsion, fecal pellet formation, absorption, and nutrient storage. Pinocytotic features and vacuoles suggest that absorption of macromolecules and intracellular digestion occur in the globular cells of the stomach and rectum. The large gastric band cells of the left lobe have typical features of intense protein synthesis and probably produce enzymes for extracellular digestion. Diffuse interdigitations of many cells enormously increase the plasmalemma surface and may be involved in liquid/ion exchange. Despite the apparent structural simplicity of the gut epithelium, O. dioica efficiently processes food to fulfill the energy requirements of its exceptionally rapid life-cycle.     

The Exceptional “Blind” Gut of Appendicularia sicula (Appendicularia, Tunicata)

In this work, we studied for the first time the histology and ultrastructure of the gut of Appendicularia sicula and demonstrated the absence of any trace of anus. Appendicularians are small holoplanktonic tunicates, characterised by very fast ingestion and quick food transfer along their gut. The high production of faecal pellets released in the aqueous environment, associated with a high filtration rate, highlights their important role in marine ecosystems. Due to the absence of an anus, in contrast with other appendicularians, A. sicula, one of the smallest species, accumulates undigested faecal material within its body, with consequent extreme enlargement of its rectum. The gut, the epithelium of which is generally extremely reduced, is formed of an oesophagus, a globular stomach, thin proximal and mid-intestine, and a huge rectum. The latter, when filled with faecal material, may occupy most of the volume of the trunk in fully grown specimens. Although profoundly altered, the gut of these animals does show several similarities to that of Fritillaria (a genus of the same family, Fritillariidae), with which it has in common many features such as specialised mitochondrial pump cells. In A. sicula, the structural simplifications of organs seem to reach their extreme condition in comparison to other appendicularians.     

Cytochemical properties of Botryllus schlosseri haemocytes: indications for morpho-functional characterisation

In the present study, we carried out a detailed light microscopy investigation of the cytochemical properties of the haemocytes of the colonial ascidian Botryllus schlosseri, using new cytochemical stains and enzymatic markers, a panel of antibodies and lectins as probes to characterise Botryllus blood cells further. Results indicate that lymphocyte-like cells are circulating undifferentiated cells recognised by anti-CD34 antibody and there are at least two defined haemocyte differentiation pathways: i) phagocytes, represented by hyaline amoebocytes and macrophage-like cells, which share similar staining properties, the same hydrolytic enzyme content as well as the presence of detectable cytochrome-c-oxidase activity, recognition by anti-CD39 and Narcissus pseudonarcissus agglutinin; ii) cytotoxic cell line, represented by granular amoebocytes and morula cells which have vacuoles stained by Ehrlich's stain and Neutral Red; DOPA-containing protein are present inside morula cell vacuoles. Pigment cells and nephrocytes are involved in catabolite storage but their relationships with other cell types are less clear.     

Oxidative stress induces cytotoxicity during rejection reaction in the compound ascidian Botryllus schlosseri

When genetically incompatible colonies of the compound ascidian Botryllus schlosseri contact each other, a rejection reaction occurs, characterised by the appearance of cytotoxic foci along the touching borders. In the course of this reaction, morula cells, a common haemocyte-type in ascidians, release their vacuolar content, mainly phenoloxidase and its polyphenol substrata, upon the recognition of soluble factors diffusing from the alien colony through the partially fused tunic. In a previous paper, we demonstrated the relationship between phenoloxidase and cytotoxicity. Here, we investigated the effects of superoxide dismutase, catalase and sorbitol (scavengers of superoxide anions, peroxides and hydroxyl radicals, respectively) on the cytotoxicity observed in haemocyte cultures incubated with heterologous blood plasma. Although the above compounds have no effects on morula cell degranulation and phenoloxidase activity, they suppress cell death, suggesting that oxidative stress plays a key role in in vitro cytotoxicity. In addition, sorbitol reduces the extent of the cytotoxicity occurring in the rejection reaction between incompatible colonies, which stresses the important role of hydroxyl radicals in this process. The observation of a decrease in total and non-protein thiols in haemocytes previously incubated with heterologous blood plasma fits the hypothesis of oxidative stress as the main cause of phenoloxidase-related cytotoxicity.     

Molecular evolution of olfactomedin

Olfactomedin is a secreted polymeric glycoprotein of unknown function, originally discovered at the mucociliary surface of the amphibian olfactory neuroepithelium and subsequently found throughout the mammalian brain. As a first step toward elucidating the function of olfactomedin, its phylogenetic history was examined to identify conserved structural motifs. Such conserved motifs may have functional significance and provide targets for future mutagenesis studies aimed at establishing the function of this protein. Previous studies revealed 33% amino acid sequence identity between rat and frog olfactomedins in their carboxyl terminal segments. Further analysis, however, reveals more extensive homologies throughout the molecule. Despite significant sequence divergence, cysteines essential for homopolymer formation such as the CXC motif near the amino terminus are conserved, as is the characteristic glycosylation pattern, suggesting that these posttranslational modifications are essential for function. Furthermore, evolutionary analysis of a region of 53 amino acids of fish, frog, rat, mouse, and human olfactomedins indicates that an ancestral olfactomedin gene arose before the evolution of terrestrial vertebrates and evolved independently in teleost, amphibian, and mammalian lineages. Indeed, a distant olfactomedin homolog was identified in Caenorhabditis elegans. Although the amino acid sequence of this invertebrate protein is longer and highly divergent compared with its vertebrate homologs, the protein from C. elegans shows remarkable similarities in terms of conserved motifs and posttranslational modification sites. Six universally conserved motifs were identified, and five of these are clustered in the carboxyl terminal half of the protein. Sequence comparisons indicate that evolution of the N-terminal half of the molecule involved extensive insertions and deletions; the C-terminal segment evolved mostly through point mutations, at least during vertebrate evolution. The widespread occurrence of olfactomedin among vertebrates and invertebrates underscores the notion that this protein has a function of universal importance. Furthermore, extensive modification of its N-terminal half and the acquisition of a C-terminal SDEL endoplasmic-reticulum- targeting sequence may have enabled olfactomedin to adopt new functions in the mammalian central nervous system.