Long term effects of chernobyl contamination on dna repair function and plant resistance to biotic and abiotic stress factors.

Thirty years after the Chernobyl explosion we still lack information regarding the genetic effects of radionuclide contamination on the plant population. For example, are plants adapting to the low dose of chronic ionising irradiation and showing improved resistance to radiation damage? Are they coping with changing/increased pathogenicity of fungi and viruses in the Chernobyl exclusion zone? Are plant populations rapidly accumulating mutational load and should we expect rapid microevolutionary changes in plants in the Chernobyl area? This review will try to summarise the current knowledge on these aspects of plant genetics and ecology and draw conclusions on the importance of further studies in the area around Chernobyl.

A national survey of support and counselling after maternal death.

The 2000-2002 Confidential Enquiry into Maternal and Child Health report highlighted several cases of maternal death where the staff who had been involved, were not offered support. The report recommended that 'Trusts must make provision for the prompt offer of support and/or counselling for all staff who have cared for a woman who has died.' We conducted a postal survey to firstly establish whether Trusts had implemented this, and also to ascertain the experience of consultant obstetric anaesthetists. Of 706 respondents (response rate 64%), 60% involved in a maternal death or other traumatic event received no offer of support, 65% were unaware of potential sources of support and only 5% received details of further help available. Furthermore, 69% were unaware of policies within their own Trusts for the provision of support services. We suggest that a formal structure should exist within all units that offers confidential support services and/or debriefing facilities to all staff involved in a maternal death or other traumatic event.

Catchment condition as a major control on the quality of receiving basin sediments (Sydney Harbour, Australia).

The current work aimed to compile existing information to better understand the source, fate and effects of metallic contaminants in one catchment-receiving basin system (Iron Cove) in Sydney Harbour (Australia). Copper, Pb and Zn concentrations of potential source materials, i.e. soils (mean 62, 410 and 340 microg g(-1), respectively) and road dust (mean 160, 490 and 520 microg g(-1), respectively) and in materials being transported to the estuary, i.e. in gully pots (mean 110, 200 and 260 microg g(-1) for Cu, Pb, and Zn, respectively), in bedload (mean 210, 880 and 1700 microg g(-1), respectively) and particulates in canals draining the catchment (mean 325, 290 and 1865 microg g(-1), respectively) were highly enriched. Estuarine sediments in the receiving basin are enriched 20 times over pre-anthropogenic concentrations and are toxic to benthic animals at the canal mouths. Stormwater remediation is required to reduce metal loads to the adjacent estuary.

Adaptation and impairment of DNA repair function in pollen of Betula verrucosa and seeds of Oenothera biennis from differently radionuclide-contaminated sites of Chernobyl.

BACKGROUND AND AIMS: The plants that have remained in the contaminated areas around Chernobyl since 1986 encapsulate the effects of radiation. Such plants are chronically exposed to radionuclides that they have accumulated internally as well as to alpha-, beta- and gamma-emitting radionuclides from external sources and from the soil. This radiation leads to genetic damage that can be countered by DNA repair systems. The objective of this study is to follow DNA repair and adaptation in haploid cells (birch pollen) and diploid cells (seed embryos of the evening primrose) from plants that have been growing in situ in different radionuclide fall-out sites in monitored regions surrounding the Chernobyl explosion of 1986. METHODS: Radionuclide levels in soil were detected using gamma-spectroscopy and radiochemistry. DNA repair assays included measurement of unscheduled DNA synthesis, electrophoretic determination of single-strand DNA breaks and image analysis of rDNA repeats after repair intervals. Nucleosome levels were established using an ELISA kit. KEY RESULTS: Birch pollen collected in 1987 failed to perform unscheduled DNA synthesis, but pollen at gamma/beta-emitter sites has now recovered this ability. At a site with high levels of combined alpha- and gamma/beta-emitters, pollen still exhibits hidden damage, as shown by reduced unscheduled DNA synthesis and failure to repair lesions in rDNA repeats properly. Evening primrose seed embryos generated on plants at the same gamma/beta-emitter sites now show an improved DNA repair capacity and ability to germinate under abiotic stresses (salinity and accelerated ageing). Again those from combined alpha- and gamma/beta-contaminated site do not show this improvement. CONCLUSIONS: Chronic irradiation at gamma/beta-emitter sites has provided opportunities for plant cells (both pollen and embryo cells) to adapt to ionizing irradiation and other environmental stresses. This may be explained by facilitation of DNA repair function.

Complex relationships between shallow muddy benthic assemblages, sediment chemistry and toxicity in estuaries in southern New South Wales, Australia.

Synoptic sediment quality triad (contaminants, benthic assemblages, toxicity testing) data were collected for sites in Sydney estuary, adjacent Cooks River and five less-modified southern estuaries. Matching data tested relationships between contaminants and benthic assemblages, correlations with specific contaminants, and the ability of sediment quality guidelines to predict the risk of adverse effects. Significant but weak relationships occurred in complex patterns between assemblages, contaminant concentrations and environmental variables. Maximum benthos abundance occurred where sediment contamination was high and was dominated by polychaetes. Spionidae (polychaete) and Galeommatidae (mollusc) abundances were strongly correlated with site environmental characteristics and with varying mixtures of metals and organic contaminants. The risk of adverse effects on benthic assemblage structure increased with increasing sediment toxicity except for areas of very high contamination and for non-bioavailable anthropogenic chemicals. The overall weight-of-evidence scores differentiated the highly modified sites from less-contaminated southern estuaries, where toxicity scores were higher than predicted.

The rad18 gene of Schizosaccharomyces pombe defines a new subgroup of the SMC superfamily involved in DNA repair.

The rad18 mutant of Schizosaccharomyces pombe is very sensitive to killing by both UV and gamma radiation. We have cloned and sequenced the rad18 gene and isolated and sequenced its homolog from Saccharomyces cerevisiae, designated RHC18. The predicted Rad18 protein has all the structural properties characteristic of the SMC family of proteins, suggesting a motor function--the first implicated in DNA repair. Gene deletion shows that both rad18 and RHC18 are essential for proliferation. Genetic and biochemical analyses suggest that the product of the rad18 gene acts in a DNA repair pathway for removal of UV-induced DNA damage that is distinct from classical nucleotide excision repair. This second repair pathway involves the products of the rhp51 gene (the homolog of the RAD51 gene of S. cerevisiae) and the rad2 gene.

A comparison of small circular DNA molecules in psi+ and psi- strains of Saccharomyces cerevisiae.

The psi+ and psi- phenotypes, which affect the efficiency of ochre suppression in yeast, are inherited in a non-Mendelian fashion. There is no apparent difference in length or in length distribution of 2 micronm circular DNA molecules between psi+ and psi- strains. It seems that the psi genetic determinant is probably not connected with the presence or absence of these small circular DNA molecules.

Isolation and characterization of a D-7 LEA protein from pollen that stabilizes glasses in vitro.

A heat-soluble protein present in substantial quantities in Typha latifolia pollen was purified to homogeneity. The protein was subjected to cyanogen bromide cleavage, and the peptides produced were separated by HPLC chromatography and sequenced. The two sequences determined were found to be related to the putative D76 LEA protein from Brassica napus seeds and one of them to the D-7 LEA protein from upland cotton. This suggests the pollen protein to be a member of the LEA group III family of proteins. The secondary structure of the protein in solution and in the dry state was investigated using Fourier transform IR spectroscopy. Whereas the protein in solution was highly unordered, being largely in a random coil conformation, the conformation was largely alpha-helical after fast drying. Slow drying reversibly led to both alpha-helical and intermolecular extended beta-sheet structures. When dried in the presence of sucrose, the protein adopted alpha-helical conformation, irrespective of drying rate. The effect of the protein on the stability of sucrose glasses was also investigated. The dehydrated mixture of sucrose and the LEA protein had higher glass transition temperatures and average strength of hydrogen bonding than dehydrated sucrose alone. We suggest that LEA proteins may play a role together with sugars in the formation of a tight hydrogen bonding network in the dehydrating cytoplasm, thus conferring long-term stability.

The in vivo conformation of the plastid DNA of Toxoplasma gondii: implications for replication.

The Phylum Apicomplexa comprises thousands of obligate intracellular parasites, some of which cause serious disease in man and other animals. Though not photosynthetic, some of them, including the malaria parasites (Plasmodium spp.) and the causative organism of Toxoplasmosis, Toxoplasma gondii, possess a remnant plastid partially determined by a highly derived residual genome encoded in 35 kb DNA. The genetic maps of the plastid genomes of these two organisms are extremely similar in nucleotide sequence, gene function and gene order. However, a study using pulsed field gel electrophoresis and electron microscopy has shown that in contrast to the malarial version, only a minority of the plastid DNA of Toxoplasma occurs as circular 35 kb molecules. The majority consists of a precise oligomeric series of linear tandem arrays of the genome, each oligomer terminating at the same site in the genetic map, i.e. in the centre of a large inverted repeat (IR) which encodes duplicated tRNA and rRNA genes. This overall topology strongly suggests that replication occurs by a rolling circle mechanism initiating at the centre of the IR, which is also the site at which the linear tails of the rolling circles are processed to yield the oligomers. A model is proposed which accounts for the quantitative structure of the molecular population. It is relevant that a somewhat similar structure has been reported for at least three land plant chloroplast genomes.

The dominant PNM2- mutation which eliminates the psi factor of Saccharomyces cerevisiae is the result of a missense mutation in the SUP35 gene.

The PNM2- mutation of Saccharomyces cerevisiae eliminates the extrachromosomal element psi. PNM2 is closely linked to the omnipotent suppressor gene SUP35 (also previously identified as SUP2, SUF12, SAL3 and GST1). We cloned PNM2- and showed that PNM2 and SUP35 are the same gene. We sequenced the PNM2- mutant allele and found a single G-->A transition within the N-terminal domain of the protein. We tested the effects of various constructs of SUP35 and PNM2- on psi inheritance and on allosuppressor and antisuppressor functions of the gene. We found that the C-terminal domain of SUP35 protein (SUP35p) could be independently expressed; expression produced dominant antisuppression. Disruption of the N-terminal domain of PNM2- destroyed the ability to eliminate psi. These results imply that the domains of SUP35p act in an antagonistic manner: the N-terminal domain decreases chain-termination fidelity, while the C-terminal domain imposes fidelity. Two transcripts were observed for SUP35, a major band at 2.4 kb and a minor band at 1.3 kb; the minor band corresponds to 3' sequences only. We propose a model for the function of SUP35, in which comparative levels of N- and C-terminal domains of SUP35p at the ribosome modulate translation fidelity.

Nuclear and mitochondrial DNA of the primate malarial parasite Plasmodium knowlesi.

Restriction analyses and DNA/DNA hybridisation of parasite DNA isolated from monkeys infected with the malarial parasite Plasmodium knowlesi has permitted unambiguous identification of the nuclear DNA of this species. Its (G+C) content, as determined by estimations of buoyant density as well as by direct analysis, is about 38%, essentially indistinguishable from that of its primate laboratory host, and grossly different from that of the major human malaria parasite, P. falciparum, which has a (G+C) content of approx. 19%. In addition, gradient fractionation of total P. knowlesi DNA revealed a minor DNA component (approx. 1% of the total) with a (G+C) content of about 19%. This DNA comprises covalently closed circular molecules which have a contour length about 11.6 microns, carry a small cruciform structure, and are thought to originate in the parasite's mitochondria.

Mitochondrial DNA of the human malarial parasite Plasmodium falciparum.

Covalently closed circular DNA molecules were isolated from Plasmodium falciparum total DNA by isopycnic centrifugation in CsCl gradients containing either ethidium bromide or 2',6-diamidino-2-phenylindole. The circular molecules had an average contour length of 11.1 +/- 0.5 micron, similar to the analogous molecules previously isolated from the simian malaria parasite P. knowlesi. Both circular molecules shared considerable sequence homology and conserved restriction sites. The nucleotide sequence of one 936 bp fragment of the P. falciparum molecule was determined and identified, by a data base homology search, as part of a mitochondrial small rRNA subunit, thus confirming the mitochondrial origin of the circular DNAs of both malarial species.

Immune-mediated congenital heart block (CHB): identifying and counseling patients at risk for having children with CHB.

OBJECTIVE: To identify patterns of maternal antibodies associated with an increased risk of having a child with congenital heart block (CHB) and to provide a basis for counseling women with a previously affected child. METHODS: This retrospective clinical study of the obstetric histories of 46 Finnish women with a CHB child compared the strength and specificity of the immune response to SS-A/Ro and SS-B/La, as determined by immunoblot and ELISA, in 44 affected women with 85 women with systemic lupus erythematosus (SLE) and 32 women with primary Sjögren's syndrome (SS) with healthy children. RESULTS: High levels of anti-SS-A/Ro and anti-SS-B/La by practically all assays were associated with a significantly increased risk of having a CHB child. The best single test to identify high-risk mothers was anti-52 kd SS-A/Ro by immunoblot (OR 18.9), and it was the only assay to detect mothers at increased risk of CHB as compared with controls with primary SS. Low risk of CHB was indicated by undetectable or low levels of antibodies in the ELISA assays and no reactivity on immunoblot. Mothers with a previous child with CHB had a history of fetal loss (mostly spontaneous abortions) or a history of recurrent fetal losses (> or = 3) slightly more often than controls. Late-trimester obstetric complications in non-CHB pregnancies were insignificant. The relative risk for a female child compared with a male child to have CHB was 1.9 (1.2-2.9, P = .009), and the risk of the mother having another child with CHB was 12% (4 of 34). CONCLUSION: Although there is no unique antibody profile specific for CHB, mothers with a high or low risk of having a child with CHB can be identified. Female children appear to have an increased risk of CHB, but the risk of the mother having another child with CHB is low.

Repair of 6-4 photoproducts and cyclobutane pyrimidine dimers in rad mutants of Saccharomyces cerevisiae.

Repair rates of both pyrimidine-pyrimidone (6-4) photoproducts and cyclobutane pyrimidine dimers have been measured in the UV-sensitive mutants of Saccharomyces cerevisiae: rad1 to rad12 and rad14 to rad24. A dot blot immunoassay for UV photoproducts was used which measures lesions in the genome as a whole and which distinguishes 6-4 photoproducts from cyclobutane dimers. The principal findings are: (1) Wild-type yeast cells, like normal mammalian cells, repair 6-4 photoproducts more rapidly than cyclobutane dimers. (2) All mutants that are defective in repair are defective in repair of both lesions. (3) The most sensitive alleles of rad1, rad2, rad3, rad4 and rad10 show no repair of either lesion. (4) Leaky alleles of rad1, rad3 and rad14 show a very marked difference in repair rates of the two lesions, rather like the human XPA revertant cell line XP129 and the Chinese hamster mutants UV61 and V-H1. (5) No mutant repairs cyclobutane dimers more rapidly than 6-4 photoproducts.

The repair of ultraviolet light-induced DNA damage in the halophilic archaebacteria, Halobacterium cutirubrum, Halobacterium halobium and Haloferax volcanii.

Extremely halophilic archaebacteria have been reported to have no capacity for dark repair (excision repair) of ultraviolet damage and to rely on very efficient photoreactivation for recovery after UVC irradiation. Post-UV incubation in the light restores 100% survival in these organisms. This has been taken to indicate that cyclobutane dimers are the only significant UV-induced lesions and that they are completely repaired by photoreactivation. However, in all organisms studied to date, pyrimidine (6-4) pyrimidone photoproducts are a significant cytotoxic and mutagenic lesion and constitute 10-30% of UV photoproducts. The question arises, therefore--are 6-4 photoproducts induced in the halophilic archaebacteria and, if they are, how are they repaired? This paper shows that both cyclobutane dimers and 6-4 photoproducts are induced in the extremely halophilic archaebacteria, Halobacterium cutirubrum, Halobacterium halobium and Haloferax volcanii, at similar levels as in other organisms. Furthermore, contrary to previous reports, there is dark repair of both lesions. As in other organisms, 6-4 photoproducts are removed more efficiently than cyclobutane dimers in the dark. In the light, cyclobutane dimers are repaired very rapidly and there is also photoenhanced repair of 6-4 photoproducts. This work confirms that organisms such as Halobacterium and Haloferax which live in conditions of high exposure to sunlight have very efficient rates of repair of UV lesions in the light.

Repair of 6-4 photoproducts in Saccharomyces cerevisiae.

We have developed a dot blot immunoassay for UV photoproducts which distinguishes 6-4 photoproducts from cyclobutane dimers. The assay uses a polyclonal antiserum that is specific for UV-irradiated DNA. Cyclobutane dimers are measured in DNA samples which have been treated with hot alkali to destroy 6-4 photoproducts. 6-4 Photoproducts are measured using blots that have been incubated in photoreactivating enzyme to eliminate cyclobutane dimers. A combination of the two treatments leaves no detectable antigenic lesions. Wild-type S. cerevisiae repairs 6-4 photoproducts, in the genome overall, more rapidly than cyclobutane dimers. The most sensitive alleles of rad1, rad2, rad3 and rad4 are completely unable to repair either kind of photoproduct. We conclude that 6-4 photoproducts are repaired by essentially the same mechanism as are cyclobutane dimers.

Repair of UV damage in the fission yeast Schizosaccharomyces pombe.

This review is concerned with repair and tolerance of UV damage in the fission yeast, Schizosaccharomyces pombe and with the differences between Sch. pombe and budding yeast, Saccharomyces cerevisiae in their response to UV irradiation. Sch. pombe is not as sensitive to ultra-violet radiation as Sac. cerevisiae nor are any of its mutants as sensitive as the most sensitive Sac. cerevisiae mutants. This can be explained in part by the fact that Sch. pombe, unlike budding yeast or mammalian cells, has an extra pathway (UVER) for excision of UV photoproducts in addition to nucleotide excision repair (NER). However, even in mutants lacking this additional pathway, there are significant differences between the two yeasts. Sch. pombe mutants that lack the alternative pathway are still more UV-resistant than wild-type Sac. cerevisiae; recombination mutants are significantly UV sensitive (unlike their Sac. cerevisiae equivalents); mutants lacking the second pathway are sensitized to UV by caffeine; and checkpoint mutants are relatively more sensitive than the budding yeast equivalents. In addition, Sch. pombe has no photolyase. Thus, the response to UV in the two yeasts has a number of significant differences, which are not accounted for entirely by the existence of two alternative excision repair pathways. The long G2 in Sch. pombe, its well-developed recombination pathways and efficient cell cycle checkpoints are all significant components in survival of UV damage.

Hypercalcemic effect of potassium iodide on serum calcium in domestic fowl.

Five experiments were conducted to determine the influence of potassium iodide (KI) on serum calcium in the laying hen. Serum calcium was significantly increased when hens were fed a diet containing 5000 p.p.m. I as KI. This occurred even though feed consumption, egg production and size of ovary and oviduct were significantly decreased. In some hens fed KI, serum calcium was increased to as high as 70 mg. %, a 163% increase. Most of the increase was in the form of non-diffusible calcium. Hens fed a diet containing 0.05% calcium had significantly reduced serum calcium values. When these calcium-deficient hens were fed 5000 p.p.m. I as KI, a significant increase in serum calcium occurred within 3 days and within 7 days their average serum calcium value was significantly greater than that of hens fed a diet containing 3.00% calcium. A combination of KI plus estradiol was significantly more effective in increasing serum calcium than was either compound alone. Although these data gave no indication as to the mechanism of action of KI on serum calcium they do offer a new model in which to study calcium metabolism in the laying hen.