Adeno-associated virus: a vector system for efficient introduction and integration of DNA into a variety of mammalian cell types.

Adeno-associated virus (AAV) is a single-stranded DNA parvovirus that is dependent on adenovirus or herpesvirus for reproductive functions. We describe the construction of recombinant AAV vectors containing the chloramphenicol acetyltransferase gene or the neomycin phosphotransferase gene. These vectors carried their respective genes into a wide variety of cell types, including primary skin fibroblasts and hematopoietic cells. Infection efficiencies varied with cell type and ranged up to 3.0%. Coinfection of two different recombinant viruses was also used to introduce two different sequences simultaneously into a given cell. Finally, methods for obtaining recombinant AAV vectors with minimal contamination of wild-type virus are described. These various attributes of AAV vectors make them a viable DNA transduction system.     

Spatial interactions between grey wolves and Eurasian lynx in Białowieża Primeval Forest, Poland

Various species of large predators are reported to influence each other through interference or exploitation competition that may affect demography and survival of the subordinate species. We analyzed spatial relationships between grey wolf (Canis lupus) and Eurasian lynx (Lynx lynx) in Białowieża Primeval Forest (BPF, eastern Poland) to determine how they partitioned the space. The wolves (n = 8) and lynx (n = 14) were radio-tracked in 1991–1999. Three wolves and seven lynx were radio-tracked simultaneously in 1994–1996. Territories of wolf packs and home ranges of lynx overlapped considerably (76% of wolf territories and 50% of lynx home ranges, on average). In three cases, their core areas were also overlapping. Wolf-lynx dyads with overlapping home ranges were simultaneously located at distances from 0 to 28 km from each other. We found neither avoidance nor attraction between wolves and lynx occupying the same areas. We concluded that in BPF, the two large predators coexist due to specialization on different preferred prey and heterogeneous habitat.     

Optimizing electroporation parameters for a variety of human hematopoietic cell lines

The parameters affecting electroporation of four human hematopoietic cell lines were investigated. The optimal conditions for electroporation are described for both transient and stable expression of foreign genes. A correlation exists between the levels of transient gene expression and stable transfection frequency. In addition, linear DNA yields higher stable transfection frequencies than supercoiled DNA. The cumulative results indicate that electroporation is a simple and useful method for obtaining transient and stable expression of foreign genes in human hematopoietic cells.     

Recombinant human acetylcholine receptor alpha-subunit induces chronic experimental autoimmune myasthenia gravis

A synthetic gene encoding the 210 N-terminal residues of the alpha-subunit of the nicotinic acetylcholine receptor (AChR) of human skeletal muscle was cloned into an inducible expression plasmid to produce a fusion protein in high yield in Escherichia coli. Like native human AChR, the recombinant human alpha 1-210 protein induced AChR-binding, AChR-modulating, and AChR-blocking autoantibodies in rats when injected once intradermally as an emulsion in CFA, with Bordetella pertussis vaccine as supplementary adjuvant. The minimum dose of recombinant protein required to induce biochemical signs of experimental autoimmune myasthenia gravis (EAMG) with 100% incidence was 2.2 micrograms. With 6.6 to 22 micrograms, serum levels of autoantibodies were persistent, and clinically apparent EAMG lasted more than a month. Clinical, electrophysiological, and biochemical indices of EAMG induced by doses of 66 micrograms or more were more uniformly severe and persistent, with 33% fatality. Rats receiving a control extract of E. coli containing plasmid without the alpha 1-210 codon insert, with adjuvants, did not develop autoantibodies or signs of EAMG. This highly reproducible new model of EAMG induced by a recombinant human autoantigen should be valuable for testing Ag-specific immunotherapeutic strategies that might be applicable to treating acquired myasthenia gravis in humans.     

Wolf predation and snow cover as mortality factors in the ungulate community of the Bialowieża National Park,Poland

Summary Wolf-ungulate interactions were studied in the pristine deciduous and mixed forests of the Bialowiea National Park in 1985–1989. The study period included two severe and two mild winters. The community of ungulates inhabiting Bialowiea National Park consisted of red deer Cervus elaphus, 55% of all ungulates; wild boar Sus scrofa, 42%; and roe deer Capreolus capreolus, moose Alces alces, and European bison Bison bonasus, about 1% each. The average size of red deer groups increased from 2.7 (SD 2.35) in spring and summer to 6.9 (SD 6.84) in autumn and winter. In winter the group size of red deer was positively correlated with the depth of snow cover and negatively correlated with the mean daily temperature. Average group size of wild boar did not change significantly between seasons; it was 6.8 (SD 5.16) in spring and summer and 5.7 (SD 4.67) in autumn and winter. Analysis of 144 wolf scats showed that wolves preyed selectively on red deer. In October–April, Cervidae (mostly red deer) constituted 91% of biomass consumed by wolves, while wild boar made up only 8%. In May–September deer formed 77% of prey biomass, and the share of wild boar increased to 22%. In all seasons of the year wolves selected juveniles from deer and boar populations: 61% of red deer and 94% of wild boar of determined age recovered from wolves' scats were young <1 year old. Analysis of 117 carcasses of ungulates found in Bialowiea National Park showed that predation was the predominant mortality factor for red deer (40 killed, 10 dead from causes other than predation) and roe deer (4 killed, none dead). Wild boar suffered most from severe winter conditions (8 killed, 56 dead). The percentage of ungulates that had died from undernutrition and starvation in the total mortality was proportional to the severity of winter.     

Concordant mitochondrial and microsatellite DNA structuring between Polish lowland and Carpathian Mountain wolves

Phylogeographic studies of highly mobile large carnivores suggest that intra-specific genetic differentiation of modern species might be the consequence of the most recent Pleistocene glaciation. However, the relative influence of biogeographical processes and subsequent human-induced population fragmentation requires a better understanding. Poland represents the western edge of relatively continuous distributions of many wide-ranging species, e.g. lynx (Lynx lynx), wolves (Canis lupus), moose (Alces alces) and, therefore, a key area for understanding historic and contemporary patterns of gene flow in central Europe. We examined wolf genetic structure in Poland and in a recently recolonized area in eastern Germany using microsatellite profiles (n = 457) and mitochondrial DNA sequencing (mtDNA, n = 333) from faecal samples. We found significant genetic structure and high levels of differentiation between wolves in the Carpathian Mountains and the Polish lowlands. Our findings are consistent with previously reported mtDNA subdivision between northern lowlands and southern mountains, and add new and concordant findings based on autosomal marker variation. Wolves in western Poland and eastern Germany showed limited differentiation from northeastern Poland. Although the presence of private alleles suggests immigration also from areas not sampled in this study, most individuals seem to be immigrants from northeastern Poland or their descendants. We observed moderate genetic differentiation between certain northeastern lowland regions separated by less than 50 km. Moreover, mtDNA results indicated a southeastern subpopulation near the border with Ukraine. The observed structure might reflect landscape fragmentation and/or ecological differences resulting in natal habitat-biased dispersal.     

Helminth parasites of an introduced invasive carnivore species, the raccoon ( Procyon lotor L.), from the Warta Mouth National Park (Poland)

We evaluated helminth parasites of the introduced North American raccoon ( Procyon lotor L.) in Poland. From June 2006 to May 2007, 91 raccoon fecal samples were collected in the Warta Mouth National Park situated in western Poland near the Polish-German border. Coprological analyses involved flotation and sedimentation. In total, 7 helminth taxa were identified in the feces: Ancylostoma spp., Baylisascaris procyonis, Capillariidae, Placoconus lotoris, Spirocerca lupi, Strongyloides procyonis, and Echinostoma sp. The parasite assemblage in the raccoon exhibited a low diversity. The results were compared with data from other European and North American studies and suggest a lower prevalence of parasites in newly invaded areas, compared with earlier invaded areas or the native range.     

The genetic structure of raccoon introduced in Central Europe reflects multiple invasion pathways

Invasions of non-native species are of great concern as they have a devastating impact on native biodiversity and can also affect the economy of a region. Multiple introductions in several locations of a new range greatly promote the success of non-native species. The raccoon (Procyon lotor) is an omnivore whose native distribution extends from southern Canada to Panama. It has been successfully introduced in many European countries. We examined the microsatellite and mitochondrial diversity of raccoon populations in Central Europe (Germany, Poland, and Czech Republic) in order to determine their introduction sources and pathways as well as the factors affecting genetic structure in this invasive species. We found low diversity of the mtDNA control region and moderate diversity of microsatellite markers. Raccoon showed three hierarchic levels of genetic structure which separate at different levels sampled from Czech Republic, Germany and raccoon inhabiting two different habitats in Poland. In Poland the raccoon population was established through migration from Germany to Czech Republic. Analysis of the intensity of migration between two different habitat types indicated source-sink dynamics in the Polish populations of raccoons. Our results confirm the high intensity of the raccoon invasion in Central Europe and point to specific measures needed as part of an effective management strategy.     

Genetic diversity and relatedness within packs in an intensely hunted population of wolvesCanis lupus

A population of grey wolvesCanis lupus Linnaeus, 1758 inhabiting Bia?owie?a Primeval Forest (BPF) on the Polish-Belarussian border has recovered after near extermination in the 1970s. Currently, it is intensively hunted in the Belarussian part of BPF and protected in the Polish part. We used a combination of molecular analysis, radiotracking, and field observation to study genetic diversity of the population after natural recolonisation and the consequences of heavy hunting for the genetic composition and social structure of wolf packs. Both microsatellite and mtDNA analyses revealed high genetic diversity. For 29 individuals and 20 microsatellite loci, the mean expected heterozygosity was 0.733. Four mtDNA haplotypes were found. Three of them had earlier been described from Europe. Their geographic distribution suggests that wolves recolonising BPF immigrated mainly from the north-east, and less effectively from the east and south-east. We traced the composition of 6 packs for a total of 26 pack-years. Packs were family units (a breeding pair with offspring) with occasional adoption of unrelated adult males, which occurred more frequently in packs living in the Belarussian part of the BPF, due to heavy hunting and poaching. Breeding pairs were half-sibs or unrelated wolves. Pair-bonds in the breeding pair lasted from 1 to 4 years and usually broke by the death of one or both mates. Successors of breeding females were their daughters, while a successor of a breeding male could be either his son or an alien wolf. As is evident from Bia?owie?a’s wolves, high genetic diversity may result from immigration of outside individuals, which are easily recruited to a heavily exploited local population.