Verbesserte Methode zur Gewinnung und Verarbeitung von freien Zellen aus kleinsten Volumina von Körperflüssigkeiten mittels Membranfiltration

Summary A cell collector for concentrating small numbers of exfoliated cells on membrane filters from minute volumes (0.05–3 ml) of body fluids and culture media is described. The apparatus incorporates a membrane pump, delivering a vacuum of 10–100 mm Hg. Filtration units (funnels and filter holders) with filtrating areas of 3, 5 and 8 mm diameter are used depending on the volume and the cell content of the fluid to be investigated. A rapid staining procedure based on the classical May-Grünwald-Giemsa technique for light microscopic observations and a modified technique for processing the cell-coated filters for electron microscopy are presented in some detail.

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Mit Unterstützung durch den Schweizerischen Nationalfonds zur Förderung der wissenschaftlichen Forschung.     

Nucleotide sequence evidence for rapid genotypic shifts in the bovine mitochondrial DNA D-loop

Mitochondrial DNA (mtDNA) is unusual in its rapid rate of evolution and high level of intraspecies sequence variation. The latter is thought to be related to the strict maternal inheritance of mtDNA, which effectively isolates within a species mitochondrial gene pools that accumulate mutations and vary independently. A fundamental and as yet unexplained aspect of this process is how, in the face of somatic and germ-line mtDNA ploidy of 10(3) to 10(5) (refs 4, 5), individual variant mtDNA molecules resulting from mutational events can come to dominate the large intracellular mtDNA population so rapidly. To help answer this question, we have determined here the nucleotide sequence of all or part of the D-loop region in 14 maternally related Holstein cows. Four different D-loop sequences can be distinguished in the mtDNA of these animals. One explanation is that multiple mitochondrial genotypes existed in the maternal germ line and that expansion or segregation of one of these genotypes during oogenesis or early development led to the rapid genotypic shifts observed.     

Anatomical, cellular and molecular analysis of 8,000-yr-old human brain tissue from the Windover archaeological site

Recovery and analysis of ancient tissue and bone of human origin has long been extensively investigated. Only recently, however, has it been technically possible to recover genetic material from ancient human and animal samples. As both previous studies involved dried tissue, it is important to determine whether other conditions may also preserve ancient tissue and genetic material. We describe here an analysis of preserved human bone and soft matter discovered in 1984-85 buried in a small swampy pond in central Florida. The recovered skeletal material represented a minimum of 40 individuals of both sexes and various ages. Corrected radiocarbon dates directly from bone and from peat matrix gave consistent ages in the range of 7,790 to 8,290 yr before present (BP). Nine individuals with intracranial soft matter were recovered and, in five of these, material recognizable as preserved or replaced brain tissue was present. Further analysis demonstrated gross anatomical features, remnant cellular structure and human DNA. As this find appears to be the oldest-known example of preserved human cell structure and DNA, it represents a significant resource for both anthropological and genetic studies.     

Inter- and intrapopulation studies of ancient humans

For a genetic analysis of ancient human populations to be useful, it must be demonstrated that the DNA samples under investigation represent a single human population. Toward that end, we have analyzed human DNA from the Windover site (7000–8000 BP). MHC-I analysis, using allele-specific oligonucleotide hybridization to PCR amplified Windover DNA, microsatellite analysis by PCR of the APO-A2 repeat and mtD-loop 3 region sequencing on multiple individuals spanning nearly the full range of estimated burial dates all confirm the hypothesis that there is a persistence of both nuclear and mitochondrial haplotypes at Windover throughout its entire period of use. Thus, Windover can be considered a single population. Neighbor-joining tree analysis of mtDNA sequences suggests that some mitochondrial types are clearly related to extant Amerind types, whereas others, more distantly related, may reflect genetically distinct origins. A more complete sequence analysis will be required to firmly resolve this issue. Calibrating genetic relationships deduced by tree analysis, radiocarbon dates and burial position, yields a human mtD-loop DNA rate of evolution of 3700 to 14,000 years per percent change. Both values are within the range of recent, independently calculated values using estimates of evolutionary divergence or theoretical population genetics. Thus we are beginning to relaize the promise of ancient DNA analysis to experimentally answer heretofore unapproachable questions regarding human prehistory and genetic change.     

Transgenerational epigenetic inheritance of longevity in Caenorhabditis elegans

Chromatin modifiers regulate lifespan in several organisms, raising the question of whether changes in chromatin states in the parental generation could be incompletely reprogrammed in the next generation and thereby affect the lifespan of descendants. The histone H3 lysine 4 trimethylation (H3K4me3) complex, composed of ASH-2, WDR-5 and the histone methyltransferase SET-2, regulates Caenorhabditis elegans lifespan. Here we show that deficiencies in the H3K4me3 chromatin modifiers ASH-2, WDR-5 or SET-2 in the parental generation extend the lifespan of descendants up until the third generation. The transgenerational inheritance of lifespan extension by members of the ASH-2 complex is dependent on the H3K4me3 demethylase RBR-2, and requires the presence of a functioning germline in the descendants. Transgenerational inheritance of lifespan is specific for the H3K4me3 methylation complex and is associated with epigenetic changes in gene expression. Thus, manipulation of specific chromatin modifiers only in parents can induce an epigenetic memory of longevity in descendants.