Microsatellite analysis for determination of the mutagenicity of extremely low-frequency electromagnetic fields and ionising radiation in vitro.

Extremely low-frequency electromagnetic fields (ELF-EMF) have been reported to induce lesions in DNA and to enhance the mutagenicity of ionising radiation. However, the significance of these findings is uncertain because the determination of the carcinogenic potential of EMFs has largely been based on investigations of large chromosomal aberrations. Using a more sensitive method of detecting DNA damage involving microsatellite sequences, we observed that exposure of UVW human glioma cells to ELF-EMF alone at a field strength of 1 mT (50 Hz) for 12 h gave rise to 0.011 mutations/locus/cell. This was equivalent to a 3.75-fold increase in mutation induction compared with unexposed controls. Furthermore, ELF-EMF increased the mutagenic capacity of 0.3 and 3 Gy gamma-irradiation by factors of 2.6 and 2.75, respectively. These results suggest not only that ELF-EMF is mutagenic as a single agent but also that it can potentiate the mutagenicity of ionising radiation. Treatment with 0.3 Gy induced more than 10 times more mutations per unit dose than irradiation with 3 Gy, indicating hypermutability at low dose.

Conservation genetics of Neotropical pollinators revisited: microsatellite analysis suggests that diploid males are rare in orchid bees

Allozyme analyses have suggested that Neotropical orchid bee (Euglossini) pollinators are vulnerable because of putative high frequencies of diploid males, a result of loss of sex allele diversity in small hymenopteran populations with single locus complementary sex determination. Our analysis of 1010 males from 27 species of euglossine bees sampled across the Neotropics at 2-11 polymorphic microsatellite loci revealed only 5 diploid males at an overall frequency of 0.005 (95% CIs 0.002-0.010); errors through genetic non-detection of diploid males were likely small. In contrast to allozyme-based studies, we detected very weak or insignificant population genetic structure, even for a pair of populations >500 km apart, possibly accounting for low diploid male frequencies. Technical flaws in previous allozyme-based analyses have probably led to considerable overestimation of diploid male production in orchid bees. Other factors may have a more immediate impact on population persistence than the genetic load imposed by diploid males on these important Neotropical pollinators.

Queen-worker conflict over male production and sex ratio in a facultatively polyandrous bumble bee, Bombus hypnorum: the consequences of nest usurpation.

Evolutionary conflicts among social hymenopteran nestmates are theoretically likely to arise over the production of males and the sex ratio. Analysis of these conflicts has become an important focus of research into the role of kin selection in shaping social traits of hymenopteran colonies. We employ microsatellite analysis of nestmates of one social hymenopteran, the primitively eusocial and monogynous bumblebee Bombus hypnorum, to evaluate these conflicts. In our 14 study colonies, B. hypnorum queens mated between one and six times (arithmetic mean 2.5). One male generally predominated, fathering most of the offspring, thus the effective number of matings was substantially lower (1–3.13; harmonic mean 1.26). In addition, microsatellite analysis allowed the detection of alien workers, those who could not have been the offspring of the queen, in approximately half the colonies. Alien workers within the same colony were probably sisters. Polyandry and alien workers resulted in high variation among colonies in their sociogenetic organization. Genetic data were consistent with the view that all males (n = 233 examined) were produced by a colony’s queen. Male parentage was therefore independent of the sociogenetic organization of the colony, suggesting that the queen, and not the workers, was in control of the laying of male-destined eggs. The population-wide sex ratio (fresh weight investment ratio) was weakly female biased. No evidence for colony-level adaptive sex ratio biasing could be detected.

Metastatic testicular carcinoma from the colon with clinical, immunophenotypical, and molecular characterization: Report of a case

We report a case of testicular metastasis from a colonic adenocarcinoma. The presentation of testicular metastasis, diagnosis, management, and possible modes of spread are reported. In addition to conventional investigations and histopathologic techniques, a molecular study of the testicular metastasis and colon primary, using microsatellite analysis, was performed to confirm the primary origin. Its potential uses are discussed.

Immunocytochemistry versus molecular fingerprinting of metastases

Examination of cytological samples of cancer to suggest a possible primary site of origin is one of the commonest and most difficult tasks of diagnostic cytopathologists. Currently, both cytomorphology and immunocytochemistry are the main approaches to this diagnostic dilemma. We report the application of microsatellite analysis in cytological samples in a patient with a primary colonic tumour and two subsequent lung nodules, which were suspected on CT scans of the chest, and compared the findings with those obtained with conventional immunocytochemistry. The molecular results were in agreement with the radiological impression and conflicted with the immunocytochemistry. We conclude that immunocytochemical and molecular biology approaches to the diagnosis of tumours may give rise to contradictory results.

The paradox of invasion: Reeves’ muntjac deer invade the British Isles from a limited number of founding females

High levels of genetic diversity and high propagule pressure are favoured by conservation biologists as the basis for successful reintroductions and ensuring the persistence of populations. However, invasion ecologists recognize the ‘paradox of invasion’, as successful species introductions may often be characterized by limited numbers of individuals and associated genetic bottlenecks. In the present study, we used a combination of high-resolution nuclear and mitochondrial genetic markers to investigate the invasion history of Reeves' muntjac deer in the British Isles. This invasion has caused severe economic and ecological damage, with secondary spread currently a concern throughout Europe and potentially globally. Microsatellite analysis based on eight loci grouped all 176 introduced individuals studied from across the species' range in the UK into one genetic cluster, and seven mitochondrial D-loop haplotypes were recovered, two of which were present at very low frequency and were related to more common haplotypes. Our results indicate that the entire invasion can be traced to a single founding event involving a low number of females. These findings highlight the fact that even small releases of species may, if ignored, result in irreversible and costly invasion, regardless of initial genetic diversity or continual genetic influx.

Donor type microchimerism is an infrequent event following liver transplantation and is not associated with graft acceptance

Donor-type microchimerism, the presence of a minority population of donor-derived haematopoietic cells following solid organ transplantation, has been postulated as a mechanism for induction of donor-specific graft tolerance. The stability, frequency, and relevance of microchimerism with respect to long-term outcome, however, remains uncertain. Using a polymerase chain reaction (PCR)-based method of microsatellite analysis of highly polymorphic short tandem repeat sequences (STRs) to detect donor-type cells, DNA from 11 patients was analyzed prospectively at specific time points for 12 months following liver transplantation, and from a further six patients retrospectively 2 years after liver transplantation. Using a panel of STRs, transient peripheral blood donor microchimerism was detected in 2 of 11 patients at a single time-point following transplantation, but persistent evidence of donor-derived cells was not observed during the study period. Analysis of DNA extracted from skin and duodenum in two patients likewise failed to show donor-type cells at these sites. None of the six patients in the retrospective arm showed donor microchimerism, resulting in an overall detection rate of 1.58%. These results suggest that donor microchimerism following liver transplantation is an infrequent event, and that the generation of graft tolerance is independent of microchimerism.

Comparative genomic analysis reveals species-dependent complexities that explain difficulties with microsatellite marker development in molluscs

Reliable population DNA molecular markers are difficult to develop for molluscs, the reasons for which are largely unknown. Identical protocols for microsatellite marker development were implemented in three gastropods. Success rates were lower for Gibbula cineraria compared to Littorina littorea and L. saxatilis. Comparative genomic analysis of 47.2?kb of microsatellite containing sequences (MCS) revealed a high incidence of cryptic repetitive DNA in their flanking regions. The majority of these were novel, and could be grouped into DNA families based upon sequence similarities. Significant inter-specific variation in abundance of cryptic repetitive DNA and DNA families was observed. Repbase scans show that a large proportion of cryptic repetitive DNA was identified as transposable elements (TEs). We argue that a large number of TEs and their transpositional activity may be linked to differential rates of DNA multiplication and recombination. This is likely to be an important factor explaining inter-specific variation in genome stability and hence microsatellite marker development success rates. Gastropods also differed significantly in the type of TEs classes (autonomous vs non-autonomous) observed. We propose that dissimilar transpositional mechanisms differentiate the TE classes in terms of their propensity for transposition, fixation and/or silencing. Consequently, the phylogenetic conservation of non-autonomous TEs, such as CvA, suggests that dispersal of these elements may have behaved as microsatellite-inducing elements. Results seem to indicate that, compared to autonomous, non-autonomous TEs maybe have a more active role in genome rearrangement processes. The implications of the findings for genomic rearrangement, stability and marker development are discussed.

DESIGN, ANALYSIS, AND RELIABILTY OF SOLAR PANEL ROTATION MECHANISM FOR MICROSATELLITE

Solar array rotation mechanism provides a hinged joint between the solar panel and satellite body, smooth rotation of the solar array into deployed position and its fixation in this position. After unlocking of solar panel (while in orbit), rotation bracket turns towards ready-to-work position under the action of driving spring. During deployment, once reached the required operating angle (defined by power subsystem engineer), the rotation bracket collides with the fixed bracket that is mounted on body of the satellite, to stop rotation. Due to the effect of collision force that may alter the rotation mechanism function, design of centrifugal brake is essential. At stoppage moment micro-switches activate final position sensor and a stopper locks the rotation bracket. Design of spring and centrifugal brake components, static finite element stress analysis of primary structure body of rotation mechanism at stoppage moment have been obtained. Last, reliability analysis of rotation mechanism is evaluated. The benefit of this study is to aid in the design of rotation mechanism that can be used in micro-satellite applications.

High-resolution organellar genome analysis of Triticum and Aegilops sheds new light on cytoplasm evolution in wheat

We have utilised polymorphic chloroplast microsatellites to analyse cytoplasmic relationships between accessions in the genera Triticum and Aegilops. Sequencing of PCR products revealed point mutations and insertions/deletions in addition to the standard repeat length expansion/contraction which most likely represent ancient synapomorphies. Phylogenetic analyses revealed three distinct groups of accessions. One of these contained all the non-Aegilops speltoides S-type cytoplasm species, another comprised almost exclusively A, C, D, M, N, T and U cytoplasm-type accessions and the third contained the polyploid Triticum species and all the Ae. speltoides accessions, further confirming that Ae. speltoides or a closely related but now extinct species was the original B-genome donor of cultivated polyploid wheat. Successive decreases in levels of genetic diversity due to domestication were also observed. Finally, we highlight the importance of elucidating longer-term evolutionary processes operating at microsatellite repeat loci.

High incidence of cryptic repeated elements in microsatellite flanking regions of galatheid genomes and its evolutionary implications

During the development of PCR primer sets for icrosatellite marker loci from enriched genomic libraries for three squat lobster species from Galatheidae (Decapoda: Anomura); Munida rugosa (Fabricius, 1775), M. sarsi (Huus, 1935), and Galathea strigosa (Linnaeus, 1761) (collectively known as squat lobsters), a number of unforeseen problems were encountered. These included PCR amplification failure, lack of amplification consistency, and the amplification of multiple fragments. Careful examination of microsatellite containing sequences revealed the existence of cryptic repeated elements on presumed unique flanking regions. BLAST analysis of these and other VNTR containing sequences (N 5 252) indicates that these cryptic elements can be grouped into families based upon sequence similarities. The unique features characterising these families suggest that different molecular mechanisms are involved. Of particular relevance is the association of microsatellites with mobile elements. This is the first reported observation of this phenomenon in crustaceans, and it also helps to explain why microsatellite primer development in galatheids has been relatively unsuccessful to date. We suggest a number of steps that can be used to identify similar problems in microsatellite marker development for other species, and also alternative approaches for both marker development and for the study of molecular evolution of species characterised by complex genome organisation. More specifically, we argue that new generation sequencing methodologies, which capitalise on parallel and multiplexed sequencing may pave the way forward for future crustacean research.

New microsatellite markers for Ulva intestinalis (Chlorophyta) and the transferability of markers across species of Ulvaceae

Macroalgal blooms are a growing environmental problem in eutrophicated coastal ecosystems. Members of the green algal genus Ulva are significant contributors to blooms, which are typically dominated by only one of several co-occurring opportunistic species. Our understanding of bloom dynamics, such as the importance of clonality, is limited because previously used genetic markers such as internal transcribed spacer sequences have shown very little resolution. Microsatellites are the marker of choice for such studies, but to date, only five primer pairs have been developed for a single member of this genus, Ulva intestinalis. We have now developed four new microsatellite markers for U. intestinalis using genome screening and restriction-ligation and tested them on individuals from six populations in the Gulf of Finland, Finland. All new markers exhibited polymorphism in U. intestinalis, with the numbers of alleles ranging from 6 to 10. On the basis of assignment tests, F-ST estimates and analysis of molecular variance, there was genetic differentiation among populations. Where significantly different, expected heterozygosity (HE) was higher than observed heterozygosity (Ho), indicating a trend toward heterozygote deficiency. This may indicate that although Ulva spores can disperse relatively efficiently, asexual reproduction can result in genetic differentiation among populations. We also tested the cross-species amplification of our primers and the five primer pairs reported previously on seven species of Ulva, Ulvaria obscura and Unbraulva olivascens (all members of the Ulvaceae). In each species, from five to nine of the loci produced an amplification product, and one to four alleles were discovered at each locus. These markers therefore have great potential for testing hypotheses about the formation and maintenance of multispecies macroalgal blooms.

Phylogeographic analysis of North American populations of the parasitic herbaceous plant Monotropa hypopitys reveals a complex history of range expansion from multiple late glacial refugia

Aim We carried out a phylogeographic study across the range of the herbaceous plant species Monotropa hypopitys L. in North America to determine whether its current disjunct distribution is due to recolonization from separate eastern and western refugia after the Last Glacial Maximum (LGM). Location North America: Pacific Northwest and north-eastern USA/south-eastern Canada. Methods Palaeodistribution modelling was carried out to determine suitable climatic regions for M. hypopitys at the LGM. We analysed between 155 and 176 individuals from 39 locations spanning the species' entire range in North America. Sequence data were obtained for the chloroplast rps2 gene (n=168) and for the nuclear ITS region (n=158). Individuals were also genotyped for eight microsatellite loci (n=176). Interpolation of diversity values was used to visualize the range-wide distribution of genetic diversity for each of the three marker classes. Minimum spanning networks were constructed showing the relationships between the rps2 and ITS haplotypes, and the geographical distributions of these haplotypes were plotted. The numbers of genetic clusters based on the microsatellite data were estimated using Bayesian clustering approaches. Results The palaeodistribution modelling indicated suitable climate envelopes for M. hypopitys at the LGM in both the Pacific Northwest and south-eastern USA. High levels of genetic diversity and endemic haplotypes were found in Oregon, the Alexander Archipelago, Wisconsin, and in the south-eastern part of the species' distribution range. Main conclusions Our results suggest a complex recolonization history for M. hypopitys in North America, involving persistence in separate eastern and western refugia. A generally high degree of congruence between the different marker classes analysed indicated the presence of multiple refugia, with at least two refugia in each area. In the west, putative refugia were identified in Oregon and the Alexander Archipelago, whereas eastern refugia may have been located in the southern part of the species' current distribution, as well as in the 'Driftless Area'. These findings are in contrast to a previous study on the related species Orthilia secunda, which has a similar disjunct distribution to M. hypopitys, but which appears to have recolonized solely from western refugia. © 2011 Blackwell Publishing Ltd.