A New Standard DNA Damage (SDD) Data Format.

Our understanding of radiation-induced cellular damage has greatly improved over the past few decades. Despite this progress, there are still many obstacles to fully understand how radiation interacts with biologically relevant cellular components, such as DNA, to cause observable end points such as cell killing. Damage in DNA is identified as a major route of cell killing. One hurdle when modeling biological effects is the difficulty in directly comparing results generated by members of different research groups. Multiple Monte Carlo codes have been developed to simulate damage induction at the DNA scale, while at the same time various groups have developed models that describe DNA repair processes with varying levels of detail. These repair models are intrinsically linked to the damage model employed in their development, making it difficult to disentangle systematic effects in either part of the modeling chain. These modeling chains typically consist of track-structure Monte Carlo simulations of the physical interactions creating direct damages to DNA, followed by simulations of the production and initial reactions of chemical species causing so-called "indirect" damages. After the induction of DNA damage, DNA repair models combine the simulated damage patterns with biological models to determine the biological consequences of the damage. To date, the effect of the environment, such as molecular oxygen (normoxic vs. hypoxic), has been poorly considered. We propose a new standard DNA damage (SDD) data format to unify the interface between the simulation of damage induction in DNA and the biological modeling of DNA repair processes, and introduce the effect of the environment (molecular oxygen or other compounds) as a flexible parameter. Such a standard greatly facilitates inter-model comparisons, providing an ideal environment to tease out model assumptions and identify persistent, underlying mechanisms. Through inter-model comparisons, this unified standard has the potential to greatly advance our understanding of the underlying mechanisms of radiation-induced DNA damage and the resulting observable biological effects when radiation parameters and/or environmental conditions change.

The mitochondrial DNA of the amoeboid protozoon, Acanthamoeba castellanii: complete sequence, gene content and genome organization.

In phylogenetic trees based on comparison of nuclear small subunit rRNA sequences, Acanthamoeba castellanii (an amoeboid protozoon) is positioned near the base of the radiation leading to the animals, fungi and plants. However, the specific affiliation of this protist with the major multicellular lineages of eukaryotes is currently uncertain. To further explore the evolutionary position of A. castellanii, we have determined the complete primary sequence of its mitochondrial genome. We find that the circular mtDNA (41,591 bp; 70.6% A+T) encodes two rRNAs (small subunit and large subunit), 16 tRNAs and 33 proteins (17 subunits of the respiratory chain and 16 ribosomal proteins). As well, this genome contains eight open reading frames (ORFs) larger than 60 codons and of undefined function. Two of these ORFs (orf124 and orf142) have homologs in other mtDNAs ("orf25" and "orfB", respectively), three are unique to A. castellanii mtDNA (orf83, orf115 and orf349), and three are intronic ORFs. Among notable features of A. castellanii mtDNA are the following: (1) Genes and ORFs are all encoded on the same strand and are tightly packed, with only 6.8% of the total sequence not having an evident coding function and intergenic spacer sequences ranging from only 1 to 616 bp (average 64 bp). Ten pairs of protein-coding genes overlap by up to 38 bp and two subunits of cytochrome oxidase (COX1 and COX2) are specified by a single continuous ORF. (2) Only three introns, all group I and each containing a free-standing ORF, are present; these are localized in the 3'-half of the large subunit rRNA gene. (3) The genome encodes fewer than the minimal number of tRNA species required to support mitochondrial protein synthesis, suggesting that additional tRNAs are imported from the cytosol into A. castellanii mitochondria. Of the 16 tRNAs specified by A. castellanii mtDNA (one with an 8-nucleotide anticodon loop), 13 have been shown or are predicted to undergo a novel form of RNA editing within the acceptor stem. (4) A modified genetic code is used in which UGA specifies tryptophan. (5) Repeated sequences and obvious small sequence motifs that might represent regulatory elements are absent. In overall size, gene content and organizational pattern, A. castellanii mtDNA most closely resembles the mtDNA of the chlorophycean alga Prototheca wickerhamii (55,326 bp; 74.2% A+T), but is quite different in these respects from the mtDNA of Chlamydomonas reinhardtii (15,758 bp; 54.8% A+T), another chlorophycean alga, as well from characterized animal and fungal mitochondrial genomes.(ABSTRACT TRUNCATED AT 250 WORDS)

Complete sequence of the mitochondrial DNA of the chlorophyte alga Prototheca wickerhamii. Gene content and genome organization.

The complete nucleotide sequence of the circular mitochondrial (mt) DNA of the chlorophyte alga Prototheca wickerhamii has been determined (55,328 base-pairs, A+T content 74.2%). The genes identified encode three subunits of the cytochome oxidase, apocytochrome b, nine subunits of the NADH dehydrogenase complex (nad1 to 7, nad4L and nad9), three ATPase subunits (atp6, atp9, atp1 (also referred to as atpA)), three ribosomal RNAs (5 S (rrn5), small subunit (srn) and large subunit (lrn) RNA), 26 tRNAs, and 13 ribosomal proteins. A total of five group I introns reside in lrn and cox1, two of which include intronic open reading frames (ORFs). Five free-standing ORFs longer than 60 codons are present. Three of these ORFs are counterparts to genes encoding proteins of unknown function in plant mitochondria (orf25 and orfB of angiosperms and orf244 of liverwort), whereas two of them are unique. Mitochondrial genes are encoded on both DNA strands in a way that suggests the existence of two transcription units, each including approximately one half of the mitochondrial genome. The two intergenic regions in which transcription is believed to initiate and terminate are about ten times longer than the other intergenic regions (1118 and 1993 nt versus 100 to 150 nt). A total of 29 recurring sequence motifs (30 to 200 nt long) have been found in intergenic regions. Nine different types of motifs are present, most of them arranged as tandem repeats. These motifs may be implicated in transcription, e.g. as signals for initiation, termination and/or processing. Phylogenetic analysis on the basis of the cox1 gene strongly suggested that P. wickerhamii and plant mitochondrial genomes are monophyletic. The finding of plant-specific mitochondrial genes such as orf25, orf244, orfB and rrn5 in P. wickerhamii mitochondria corroborates this idea.

Simulation of the radiolysis of water using Green's functions of the diffusion equation.

Radiation chemistry is of fundamental importance in the understanding of the effects of ionising radiation, notably with regard to DNA damage by indirect effect (e.g. damage by ·OH radicals created by the radiolysis of water). In the recent years, Green's functions of the diffusion equation (GFDEs) have been used extensively in biochemistry, notably to simulate biochemical networks in time and space. In the present work, an approach based on the GFDE will be used to refine existing models on the indirect effect of ionising radiation on DNA. As a starting point, the code RITRACKS (relativistic ion tracks) will be used to simulate the radiation track structure and calculate the position of all radiolytic species formed during irradiation. The chemical reactions between these radiolytic species and with DNA will be done by using an efficient Monte Carlo sampling algorithm for the GFDE of reversible reactions with an intermediate state that has been developed recently. These simulations should help the understanding of the contribution of the indirect effect in the formation of DNA damage, particularly with regards to the formation of double-strand breaks.

Binary-Encounter-Bethe ionisation cross sections for simulation of DNA damage by the direct effect of ionising radiation.

DNA damage is of crucial importance in the understanding of the effects of ionising radiation. To refine existing DNA damage models, an approach using the Binary-Encounter-Bethe (BEB) cross sections was developed. The differential cross sections for ionisation of the molecular orbitals of the DNA bases, sugars and phosphates are calculated using the electron binding energy, the mean kinetic energy and the occupancy number of each orbital as parameters. The resulting cross section has an analytic form which is quite convenient to use for Monte-Carlo codes that randomly sample the energy loss occurring during an ionisation event. We also describe an algorithm to simulate the interactions of electrons with DNA in the radiation transport code RITRACKS using the integrated BEB cross section for the bases, sugar and phosphates.

Evaluation for Hsp70 as a biomarker of effect of pollutants on the earthworm Lumbricus terrestris.

Induction of heat shock proteins (Hsps) is often associated with a cellular response to a harmful stress or to adverse life conditions. The main aims of the present study were (1) to assess if stress-induced Hsp70 could be used to monitor exposure of the earthworm species Lumbricus terrestris to various soil pollutants, (2) to assess the specificity of pollutants in their tissue targeting and in Hsp70 induction, and (3) to evaluate if dose-response relationships could be established and if the stress-response observed was specific. The midgut/intestinal tissues of L. terrestris are shown to express an inducible member of the Hsp70 family after heat shock treatment in vitro and exposures to different soil toxicants in vivo (re: artificial soil). Short-term (24-72 hours) and long-term (14-16 days) exposures to the chemical standards chloroacetamide and pentachlorophenol and to heavy metals (Pb++, Cd++, Cu++, and Hg++) also affected the earthworms, and Hsp70 was induced in their midgut/intestinal tissues. After a 3-day exposure to heavy metals, the level of Hsp70 induction in the midgut/intestinal tissues appears to correlate well with the reported in vivo and in vitro toxicity data. Comparatively, in proximal and midbody wall muscle tissues of animals exposed to the heavy metals, a decrease in expression of Hsp70 was sometimes detected. Thus Hsp analysis by Western blot in L. terrestris tissues and particularly in the midgut/intestine proved to be a suitable and sensitive assay for adverse effects in earthworms and showed a good level of reproducibility despite some individual variations. The use of pristine/nonexposed animals transposed into contaminated environments as in the present study should therefore be of high ecological relevance. Induction of Hsp70 in earthworms should represent not only a good wide-spectrum biomarker of exposure but also a biomarker of effect since known toxicants altered gene expression in tissues of these animals, as contrasted with a simple accumulation of Hsp. Hence, the detection of Hsp70 in earthworms can constitute an early-warning marker for the presence of potentially deleterious agents in soils, with L. terrestris in particular and earthworms in general acting as potential sentinel animal species.

Dietary composition alters gentamicin-induced nephrotoxicity in rats.

Previous studies have shown temporal variations in gentamicin-induced renal toxicity characterized by a peak when administered during the resting period and a trough during the active period. This time-dependent toxicity was also altered according to the macronutrient composition of dietary regimens offered to female rats. In the present study, adult female Sprague-Dawley rats were adapted to semipurified isocaloric diets containing 20% casein or soy-protein (10% fat each) or to a standard chow diet (18.1% mixed proteins; 4.5% fat). The animals were then chronically treated for 10 days with a nephrotoxic dose of gentamicin sulfate (40 mg/kg/day ip) or a saline solution administered in the middle of their resting period (1200 h) or in the middle of their activity period (0000 h). Body weights of rats injected in the middle of their resting period decreased over the last 6 days of gentamicin treatment. Total 12-h light and 12-h dark food intakes were decreased in gentamicin-treated rats. Rats fed the standard chow diet had significantly lower corticocellular regeneration, serum creatinine and blood urea nitrogen compared to those fed the casein- and soy-containing diets. The present study demonstrates that chronic gentamicin-induced renal toxicity varies temporally according to the time of administration and that a mixed protein diet containing a lower fat level can protect against gentamicin-induced nephrotoxicity.

Cx43 suppresses mammary tumor metastasis to the lung in a Cx43 mutant mouse model of human disease.

Gap junctions, the channels formed by the connexin (Cx) family of proteins, are responsible for direct intercellular communication. Although connexins are considered as tumor suppressors, their overall role in cancer onset, progression and metastasis is somewhat controversial. This study uses a novel Cx43 mutant mouse model (G60S mice) and cross-breeding strategies to determine the role of Cx43 in all stages of breast tumorigenesis. G60S mice were cross-bred with ErbB2 overexpressing mice, and spontaneous and 7,12-dimethylbenz[α]anthracene (DMBA)-induced tumor development was evaluated. Mice were killed when tumors reached ∼1 cm(3) or when mice showed signs of critical illness. In both spontaneous and DMBA studies, onset of palpable tumors was delayed in G60S mice compared with mice in control groups. Moreover, while tumors from control mice reached the size threshold, most DMBA-exposed Cx43 mutant mice were killed prematurely because of labored breathing, independent of the presence of a palpable tumor. Reduced Cx43 levels in Cx43 mutant mice were accompanied by extensive mammary gland hyperplasia. Lung histology revealed that all Cx43 mutant mice exhibited mammaglobin-positive mammary gland metastases to the lung, and the number of metastases was increased by threefold in Cx43 mutant mice on treatment with DMBA. Thus, while reduced levels of Cx43 delayed the onset of palpable tumors, normal Cx43 levels inhibited mammary gland tumor metastasis to the lungs. Understanding the mechanisms of how Cx43, which is expressed primarily in myoepithelial cells, inhibits mammary gland tumor metastasis is critical as Cx43 is assessed as a candidate for therapeutic intervention.

The complete mitochondrial DNA sequences of Nephroselmis olivacea and Pedinomonas minor. Two radically different evolutionary patterns within green algae.

Green plants appear to comprise two sister lineages, Chlorophyta (classes Chlorophyceae, Ulvophyceae, Trebouxiophyceae, and Prasinophyceae) and Streptophyta (Charophyceae and Embryophyta, or land plants). To gain insight into the nature of the ancestral green plant mitochondrial genome, we have sequenced the mitochondrial DNAs (mtDNAs) of Nephroselmis olivacea and Pedinomonas minor. These two green algae are presumptive members of the Prasinophyceae. This class is thought to include descendants of the earliest diverging green algae. We find that Nephroselmis and Pedinomonas mtDNAs differ markedly in size, gene content, and gene organization. Of the green algal mtDNAs sequenced so far, that of Nephroselmis (45,223 bp) is the most ancestral (minimally diverged) and occupies the phylogenetically most basal position within the Chlorophyta. Its repertoire of 69 genes closely resembles that in the mtDNA of Prototheca wickerhamii, a later diverging trebouxiophycean green alga. Three of the Nephroselmis genes (nad10, rpl14, and rnpB) have not been identified in previously sequenced mtDNAs of green algae and land plants. In contrast, the 25,137-bp Pedinomonas mtDNA contains only 22 genes and retains few recognizably ancestral features. In several respects, including gene content and rate of sequence divergence, Pedinomonas mtDNA resembles the reduced mtDNAs of chlamydomonad algae, with which it is robustly affiliated in phylogenetic analyses. Our results confirm the existence of two radically different patterns of mitochondrial genome evolution within the green algae.

Genome structure and gene content in protist mitochondrial DNAs.

Although the collection of completely sequenced mitochondrial genomes is expanding rapidly, only recently has a phylogenetically broad representation of mtDNA sequences from protists (mostly unicellular eukaryotes) become available. This review surveys the 23 complete protist mtDNA sequences that have been determined to date, commenting on such aspects as mitochondrial genome structure, gene content, ribosomal RNA, introns, transfer RNAs and the genetic code and phylogenetic implications. We also illustrate the utility of a comparative genomics approach to gene identification by providing evidence that orfB in plant and protist mtDNAs is the homolog of atp8 , the gene in animal and fungal mtDNA that encodes subunit 8 of the F0portion of mitochondrial ATP synthase. Although several protist mtDNAs, like those of animals and most fungi, are seen to be highly derived, others appear to be have retained a number of features of the ancestral, proto-mitochondrial genome. Some of these ancestral features are also shared with plant mtDNA, although the latter have evidently expanded considerably in size, if not in gene content, in the course of evolution. Comparative analysis of protist mtDNAs is providing a new perspective on mtDNA evolution: how the original mitochondrial genome was organized, what genes it contained, and in what ways it must have changed in different eukaryotic phyla.