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PubMed2000

Genetic structure and AFLP variation of remnant populations in the rare plant Pedicularis palustris (Scrophulariaceae) and its relation to population size and reproductive components.

We investigated plant reproduction in relation to genetic structure, population size, and habitat quality in 13 populations of the rare biennial plant Pedicularis palustris with 3-28500 flowering individuals. We used AFLP (amplified fragment length polymorphism) profiles to analyze genetic similarities among 129 individuals (3-15 per population). In a cluster analysis of genetic similarities most individuals (67%) were arranged in population-specific clusters. Analysis of molecular variance indicated significant genetic differentiation among populations and among and within subpopulations (P < 0.001). Gene flow (N(e) m) was low (0.298). On average, plants produced 55 capsules, 17 seeds per fruit, and 42 seedlings in the following growing season. The number of seeds per capsule was independent of population size and of genetic variability. In contrast, the number of capsules per plant (P < 0.05) and the number of seedlings per plant (P < 0.05) were positively correlated with population size. The relation between population size and the number of seeds per plant was not significant (P = 0.075). The number of capsules and of seeds and seedlings per plant (P < 0.01) were positively correlated with genetic variability. Genetic variability was independent of actual population size, suggesting that historical population processes have to be taken into account, too. Stepwise multiple regressions revealed additional significant relationships of habitat parameters (soil pH, C:N ratio), vegetation composition, and standing crop on reproductive components. We conclude that populations of P. palustris are genetically isolated and that reproductive success most likely is influenced by population size, genetic variability, and habitat quality. Management strategies such as moderate grazing, mowing, and artificial gene flow should endeavor to increase population size as well as genetic variation.

Journal Article↗
PubMed1992

Immunoglobulin VH usage analysis by fluorescent in situ hybridization and flow cytometry.

We have devised a flow cytometry-based fluorescent in situ hybridization assay that permits analysis of gene expression in a large number of single cells. In this technique, fixed and permeabilized cells are incubated with biotinylated single-stranded RNA probes and by means of a fluorescently labelled second-step reagent, the cells are analyzed by flow cytometry. This is a rapid and simple method that allows all of the steps in the procedure to be performed on cells in suspension. Using this approach, we demonstrate here that immunoglobulin heavy chain variable region (VH) gene expression can be analyzed among individual cells using particular VH family-specific probes. This technique has a high degree of accuracy (greater than 97%) in detecting the fraction of cells expressing a specific message in a population and is sensitive enough to detect immunoglobulin message in LPS activated B cells. The technique has been applied successfully to monitor gene expression in homogeneous and heterogeneous populations. It also allows concurrent analysis of cell surface proteins and gene expression through two-color flow cytometry. This method of monitoring gene expression in individual cells may have a number of applications in immunology and cell biology.

Animals↗
PubMed2004

Ecological determinants of genetic diversity in an expanding population of the shrub Myrica cerifera.

The ecological mechanisms that contribute to the acquisition of genetic diversity in an expanding population of the shrub, Myrica cerifera, on an island habitat were investigated. Genealogical reconstruction was used to assess the contribution of early reproductive colonists to subsequent recruitment. In addition, through determination of parentage, the source of recruiting seedlings was identified and the contribution of seed and pollen dispersal into the colonizing sites was inferred. The relative contribution of different sources of gene flow was determined directly and an investigation was made into how variability in breeding patterns may have contributed to observed levels of genetic variability. It was expected that early colonists that could flower would contribute to subsequent recruiting cohorts, and that the limited number of such early reproductive colonists would lead to variance in mating success, inbreeding, or bottlenecks which could reduce genetic diversity and increase genetic differentiation among subsequent recruiting cohorts. Analyses of parentage (with paternity exclusion probability > 95%) for all recruiting plants demonstrated that in fact, there was little contribution by the early reproductive colonists to subsequent cohorts, and that immigration from outside the study sites in the form of seed dispersal accounted for over 94% of the recruitment in the study plots, with pollen dispersal accounting for less than 3% gene flow. No genetic bottleneck or evidence of reproductive skew in the recruiting cohorts were found, suggesting that propagule dispersal was from many source individuals in other established populations.

Gene Frequency↗
PubMed2001

Microsatellites reveal high levels of gene flow among populations of the California squid Loligo opalescens.

Information on the extent of genetic differentiation among populations of the squid Loligo opalescens is crucial for the conservation of this commercially utilized species. We analysed six highly variable microsatellite loci in 11 collections of L. opalescens from different locations and spawning seasons to estimate the relative influence of two major evolutionary forces, gene flow and genetic drift. Microsatellite allele frequency patterns suggest that gene flow prevents population differentiation in L. opalescens. Tests for genetic differentiation showed homogeneity of the samples with an overall FST/RST of 0.0028/-0.0013. Genetic uniformity among samples from different year classes indicates that allele frequency patterns in L. opalescens are relatively stable over time. However, a more complete and detailed picture of fine-scale allele frequency shifts in this species will require a systematic microsatellite analysis of local populations over consecutive spawning cycles.

Animals↗
PubMed1993

Production and fluorescence-activated cell sorting of Escherichia coli expressing a functional antibody fragment on the external surface.

We have expressed a single chain Fv (scFv) antibody fragment, consisting of the variable heavy and variable light domains from two separate anti-digoxin monoclonal antibodies, on the external surface of Escherichia coli by fusing it to an Lpp-OmpA hybrid previously shown to direct heterologous proteins to the cell surface. This scFv fusion was expressed at a high level and was shown to bind the hapten with high affinity and specificity. Whole cell ELISAs, fluorescence microscopy, protease sensitivity, and flow cytometry all confirmed that the scFv was anchored on the outer membrane and was accessible on the surface. Utilizing fluorescence-activated cell sorting, we were able to specifically enrich scFv-producing cells from a 10(5)-fold excess of control cells in only two steps. The expression of antibody fragments on the surface of E. coli is being evaluated as an attractive method for the in vitro production and selection of useful antibody fragments.

Amino Acid Sequence↗
PubMed2003

Characterization of courtship sounds of species of the subgroup fasciola (Diptera, Drosophilidae, Drosophila repleta group): interspecific and interpopulational analyses.

The aim of this work was to characterize the male courtship song pattern of various species of the fasciola subgroup and to determine the level of variation both within and among species. The parameters analyzed were intrapulse interval (PI), interpulse interval (IPI), and intrapulse frequency (IF). Six different species were analyzed: D. coroica (three populations), D. ellisoni, D. fascioloides, D. moju, D. onca, and D. rosinae (one population each). There were significant differences among the six species for these three courtship song parameters. The IPI was the most variable parameter among these species, suggesting that this parameter is important for female discrimination. Four different hypotheses could explain this variation: 1. different selection pressures with absence of flow gene; 2. intraspecific sexual selection; 3. sympatric effects on song evolution; and 4. genetic drift. The PI was the only parameter that was significantly different among the three population of D. coroica. Low variability among populations within the same species was already observed for other subgroups and could be explained by the following hypotheses: strong selection acting on the song parameters, gene flow, or recent colonization from a common source. Additional studies of the courtship song of other species of the fasciola subgroup, as well as for other subgroups of the repleta group, and studies, using molecular makers, that focus on the genetic basis of the differences among these species in courtship song would allow us to evaluate the association of courtship song and sexual isolation in these species, and would also help us to understand the evolution of these behavioural differences.

Animals↗
PubMed1996

The V4-34 encoded anti-i autoantibodies recognize a large subset of human and mouse B-cells.

Autoantibodies to the i, I and Pr2 carbohydrate determinants bind red blood cells, preferentially at low temperature in vitro. Using multiparameter flow cytometric analyses, we demonstrate that each of these autoantibodies also react with human and mouse lymphocytes at physiologic temperatures. The anti-Pr2 autoantibody recognizes a glycoprotein determinant(s) expressed by a subset of both T and B lymphocytes. In contrast, the binding of anti-i and anti-I antibodies each is restricted to B-lymphocytes. The anti-i autoantibody binds to over 50% of all B cells, whereas the anti-I antibody reacts with less than 10% of either tonsillar or blood B cells. Prior studies identified that the B cell isoform of CD45 (B220) has the linear poly-N-acetyllactosamine that forms the "i" determinant. Because anti-B220 antibodies recently have been reported to influence T-dependent B-cell isotype switching, we tested each antibody for its ability to influence the production of secondary Ig isotypes by murine splenocytes co-cultured with a stimulator helper T cell clone. We find that addition of anti-i antibody increases the proportion of B cells secreting secondary Ig isotypes. In contrast, the anti-I antibody had no such effect. These findings imply that stimulation of B cells through the highly conserved carbohydrate determinant that forms the "i" antigen may be of physiologic importance in T-dependent B-cell differentiation.

Animals↗
PubMed2004

Humanization of agonistic anti-human 4-1BB monoclonal antibody using a phage-displayed combinatorial library.

Given the key role 4-1BB plays in the stimulation of T cells, humanization of agonistic anti-human 4-1BB monoclonal antibody (mAb) may have important clinical applications. In this paper, we present the humanization of agonistic anti-human 4-1BB mAb, BBK-4, using a phage display library. We first prepared the combinatorial library by incorporating murine and human alternative at positions representing buried residues that might affect the structural integrity of the antigen binding site. Six humanized single chain Fv (scFv) fragments were selected from the combinatorial library expressing phage-displayed humanized scFv. They were found to retain the epitope specificity of the original mAb but had affinities of lower than 1/10 of the original. In spite of the lower affinity, the humanized scFv coated on the surface expanded human peripheral blood mononuclear cells (PBMCs) in MLR similarly to the original mAb in the presence of anti-CD3 mAb. These results suggest that humanized anti-human 4-1BB scFvs can be used as a valuable reagent for clinical application.

Amino Acid Sequence↗
PubMed1999

Isolation and characterization of an anti-CD16 single-chain Fv fragment and construction of an anti-HER2/neu/anti-CD16 bispecific scFv that triggers CD16-dependent tumor cytolysis.

Bispecific antibody (bsAb)-based clinical trials of cancer have been conducted primarily using intact murine monoclonal antibody (mAb)-derived molecules. In some of these trials, toxicity resulting from the interactions of antibody Fc domains with cellular Fc receptors has limited the doses of antibody (Ab) that can be employed. Furthermore, human anti-mouse Ab responses prohibit multiple therapy courses. These factors have decreased the efficacy of the bsAb 2B1, which targets the extracellular domains (ECD) of the HER2/neu protooncogene product and the human FcgammaRIII (CD16). To address these obstacles, we have constructed and characterized a fully human gene-fused bsAb from single-chain Fv (scFv) molecules specific for HER2/neu and CD16. The human anti-CD16 scFv component, NM3E2, was isolated from a human scFv phage display library. As binding of NM3E2 to human neutrophil-associated CD16 decreased in the presence of plasma IgG, we have concluded that NM3E2 recognizes an epitope in the vicinity of the Fc binding pocket. Furthermore, the NM3E2 scFv was found by surface plasmon resonance-based epitope mapping to share an overlapping epitope with the Leu-11c mAb. The human anti-HER2/neu scFv component, C6.5, which was previously isolated from a human scFv phage display library, was employed as fusion partner for the creation of a bispecific scFv (bs-scFv). In the presence of the C6.5 x NM3E2 bs-scFv, peripheral blood lymphocytes promoted significant lysis of human SK-OV-3 ovarian cancer cells overexpressing HER2/neu. Biodistribution studies performed in SK-OV-3 tumor-bearing scid mice revealed that 1% ID/g of 125I-labeled C6.5 x NM3E2 bs-scFv was specifically retained in tumor at 23 h following injection. These results indicated that both scFv components of the bs-scFv retained their function in the fusion protein. This bsAb should overcome some of the problems associated with the 2B1 bsAb. C6.5 x NM3E2 bs-scFv offers promise as a platform for multifunctional binding proteins with potential clinical applications as a result of its human origin, lack of an Fc domain, ease of production, high level of in vitro tumor cell cytotoxicity and highly selective tumor targeting.

Animals↗
PubMed2006

Evidence of influenza a virus RNA in siberian lake ice.

Influenza A virus infects a large proportion of the human population annually, sometimes leading to the deaths of millions. The biotic cycles of infection are well characterized in the literature, including in studies of populations of humans, poultry, swine, and migratory waterfowl. However, there are few studies of abiotic reservoirs for this virus. Here, we report the preservation of influenza A virus genes in ice and water from high-latitude lakes that are visited by large numbers of migratory birds. The lakes are along the migratory flight paths of birds flying into Asia, North America, Europe, and Africa. The data suggest that influenza A virus, deposited as the birds begin their autumn migration, can be preserved in lake ice. As birds return in the spring, the ice melts, releasing the viruses. Therefore, temporal gene flow is facilitated between the viruses shed during the previous year and the viruses newly acquired by birds during winter months spent in the south. Above the Arctic Circle, the cycles of entrapment in the ice and release by melting can be variable in length, because some ice persists for several years, decades, or longer. This type of temporal gene flow might be a feature common to viruses that can survive entrapment in environmental ice and snow.

Base Sequence↗
PubMed1978

[Gene constitution of miniature "shiba" goats (author's transl)].

Genetic surveys were carried out on the miniature "Shiba" goats which have been raised in Nagasaki Prefecture, Japan. Recently, in thhe prefecture the number of "Shiba" goats have been markedly decreased to totally about 190 which are kept now only in two towns. Morphological genetic traits were observed to have been coming homogenous in Nagasaki Prefecture, and homogeneity of the traits in the colonies kept the National Institute of Animal Industry and in Stock Farm of University of Tokyo, especially in the latter, was remarkably high. The "Shiba" goats have white coat color, horns and supernumerary teats, but not wattles. Electrophoretic examinations of genetic variations at 27 blood protein loci revealed that variability in the "Shiba" goat populations were lower than that of the Japanese Saanen breed, and that the amount of gene flow from the Saanen breed into the "Shiba" goats was estimated to be smaller than into the present-day Okinawa meat goats. The genetic variability of the colony in Stock Farm of University of Tokyo was observed to be conspicuously low. From the results of pedigree analysis such a decay of genetic variability was postulated to come about from unavoidable inbreeding resulted from smallness both in numbers of foundation animals and in effective population size of the colony.

Animals↗
PubMed2006

Comparison of bone marrow and peripheral blood ZAP-70 status examined by flow cytometric immunophenotyping in patients with chronic lymphocytic leukemia.

BACKGROUND: : The mutational status of the immunoglobulin heavy chain variable gene in patients with chronic lymphocytic leukemia correlates with prognosis. Patients with mutated IgVH genes fare better than those with unmutated genes. Gene expression profiling studies identified the tyrosine kinase ZAP-70 to be expressed in unmutated CLL samples. Flow cytometric examination of ZAP-70 expression in tumor cells has been proposed to be a convenient surrogate marker for IgVH mutational status. However, a few studies have shown a small number of discordant results between ZAP-70 positivity, IgVH mutational status, and clinical outcome. There have been no reported studies comparing bone marrow samples with peripheral blood for ZAP-70 expression in CLL patients. METHODS: : We searched our flow cytometry files from October 2004 through April 2006 and identified CLL in 311 bone marrow and peripheral blood specimens from 256 patients. We defined ZAP-70 positivity as greater than 30% of the CD19(+) B-cells above the isotype control value that coexpress ZAP-70. Statistical analyses were performed using the Fisher exact test and student t-test. RESULTS: : A significantly greater number of bone marrow specimens were positive for ZAP-70 when compared with the number of peripheral blood specimens. Of all the ZAP-70 negative specimens, CLL cells from bone marrow had a greater mean percentage of ZAP-70 positive cells when compared with the CLL cells from peripheral blood. Finally, six patients were identified who were ZAP-70 positive in the bone marrow but ZAP-70 negative in the peripheral blood. CONCLUSIONS: : These results may be due to either an increase in the false positive rate in bone marrow specimens or to an intrinsic feature of CLL cells in the compartment that is biologically distinct from peripheral tumor cells. As prognosis and treatment decisions may be based on ZAP-70 results from either specimen type, it is prudent to further examine this observation. (c) 2006 International Society for Analytical Cytology.

Adult↗
PubMed2001

Comparative allozyme and microsatellite population structure in a narrow endemic plant species, Centaurea corymbosa Pourret (Asteraceae).

Centaurea corymbosa Pourret (Asteraceae) is a narrow endemic species known only from six populations located in a 3-km2 area in the south of France. Earlier field experiments have suggested that pollen and seed dispersal were highly restricted within and among populations. Consistent with the field results, populations were highly differentiated for five allozyme loci and among-population variation fitted an isolation-by-distance model. In the present study, we investigated the genetic structure of C. corymbosa using six microsatellite loci. As with allozymes, microsatellites revealed no within-population structure and a large differentiation among populations. However, allozyme loci were less powerful than microsatellites in detecting the extent of gene flow assessed by assignment tests. The patterns of structuration greatly varied among loci for both types of marker; we suggest that differences in single-locus pattern could mainly be an effect of stochastic variation for allozymes and an effect of variation in mutation rate for microsatellites. In contrast to the multilocus results, the two most polymorphic microsatellite loci did not show any isolation-by-distance pattern. Our results suggest that highly variable loci might not always be the best suited markers to quantify levels of gene flow among populations.

Asteraceae↗
PubMed1975

Population structure, gene flow and natural selection in populations of Euphydryas phaeton.

An examination of seven proteins, presumably encoded by seven structural gene loci, in three local populations of the supposedly sedentary and colonial butterfly, Euphydryas phaeton revealed that three (43 per cent) were polymorphic with three to five alleles each. In addition to this high level of heterozygosity, no statistically significant differences in allele frequencies were found at two of the three polymorphic loci. Since the effective breeding size in each population was estimated to range from as few as 20 to 200 individuals, it appears that some level of gene flow between populations must be invoked to explain the high levels of genetic variability maintained in local populations of this butterfly, despite its apparently colonial nature.

Alleles↗
PubMed2003

Genetic structure of Mediterranean populations revealed by Y-chromosome haplotype analysis.

The allelic variability at six Y-chromosome-specific polymorphisms (YAP, DYS19, DYS389-I, DYS390, DYS391, and DYS392) was used to generate male-specific haplotypes in 333 males representing 12 population samples from the region around the Mediterranean sea. Extreme interindividual variation was observed, as more than 160 distinct Y-chromosome variants could be defined as six-locus haplotypes. Concomitant with this high variability, low levels of population genetic structure were observed. In particular, a "core" of populations directly facing the north and the east of the Mediterranean basin, from the Middle East to the Italian Peninsula, was found to be genetically undifferentiated. This observation, supported by a reanalysis of Y-specific binary polymorphisms in the same populations, suggests that at least part of the male-specific gene pools of these populations has either a very recent common origin (that could be related with the Neolithic demic diffusion hypothesis), and/or that gene flow has played a significant role in shaping the patterns of genetic variability in this region. In agreement with both hypotheses, we found that the spatial distribution of DYS392 alleles revealed a marked differentiation between the East and the West of the Mediterranean area. Through the analysis of microsatellite variation, the time to the most recent common ancestor (TMRCA) of the YAP(+) sublineage 4 has been estimated. The estimations, based on two different data sets, turn out to be quite recent (7,000-11,000 YBP), suggesting that this lineage may have been first introduced into Southern Europe through Neolithic migrations from the Middle East.

Chromosomes, Human, Y↗
PubMed2004

Fine-scale geographical structure, intra-individual polymorphism and recombination in nuclear ribosomal internal transcribed spacers in Armeria (Plumbaginaceae).

BACKGROUND AND AIMS: Isolation and drift are the main causes for geographic structure of molecular variation. In contrast, the one found in a previous survey in Armeria (Plumbaginaceae) for nuclear ribosomal ITS multicopy regions was species-independent and has been hypothesized to be due to extensive gene-flow and biased concerted evolution. Since this was inferred from a genus-level phylogenetic analysis, the aim of this study was to check for the occurrence of such structure and the validity of the proposed model at a local scale, in a southern Spanish massif (Sierra Nevada), as well as to examine the evolutionary implications at the organism level. METHODS: In addition to 117 sequences of direct PCR products from genomic DNA, 50 sequences of PCR products from cloned DNA were obtained to analyse cases of intragenomic polymorphisms for the ITS regions. KEY RESULTS: Sequence data confirm the occurrence of a species-independent structure at a local scale and reveal insights through the analysis of contact areas between different ITS copies (ribotypes). A comparison between cloned and direct sequences (a) confirms that, within these contact areas, ITS copies co-occur both in different individuals and within single genomes; and (b) reveals recombination between different copies. CONCLUSIONS: This study supports the utility of direct sequences for detecting intra-individual polymorphism and for partially inferring the ITS copies involved, given previous knowledge of the variability. The main evolutionary implication at the organism level is that gene-flow and concerted evolution shape the geographic structure of ITS variation.

Base Sequence↗
PubMed1993

Long-distance gene flow in a cooperative breeder detected in genealogies of mitochondrial DNA sequences.

Rates of gene flow among populations of cooperatively breeding birds have been inferred primarily from distributions of observed distances of dispersal from birthplace to place of first breeding. However, for most cooperative breeders, characteristics of geographic population structure and extent of realized gene flow as measured by genetic markers are not known. To estimate rates of gene flow in a cooperatively breeding bird, I conducted a continent-wide survey of DNA sequence diversity in the most variable part of the mitochondrial (mt) genome among 163 grey-crowned babblers (Pomatostomus temporalis) throughout Australia and Papua New Guinea. The variability found among 86 types of mtDNA was used to infer genealogical trees relating sequences within both eastern (P. t. temporalis) and western (P. t. rubeculus) babblers. The genealogies imply low but detectable levels of gene flow between populations separated by over 1000 km. In addition, they suggest that genes from unrelated migrants contribute to genetic diversity in 5 of 50 (10%) social groups, occasionally producing large (> 3%) intragroup sequence differences. However, on average, the fraction of sequence diversity apportioned among populations (Fst) and among social groups within populations (Fgs), was substantial for both P. t. temporalis and P t. rubeculus, implying a large opportunity for the spread of social behaviours in both lineages of babblers.

Animals↗
PubMed2004

Population structure and speciation in the genus Tursiops based on microsatellite and mitochondrial DNA analyses.

Bottlenose dolphins (Tursiops truncatus) have a world-wide distribution, and show morphotypic variation among regions. Distinctions between coastal and pelagic populations have been documented; however, regional patterns of differentiation had not been previously investigated in a wider geographic context. We analysed up to nine different populations from seven different areas of the world by mitochondrial DNA and microsatellite DNA markers, and found differentiation among all putative regional populations. Both mtDNA and microsatellite DNA data show significant differentiation, suggesting restricted gene flow for both males and females. Dolphins in coastal habitat showed less variability and were in most cases differentiated from a pelagic lineage, which could suggest local founder events in some cases. Two coastal populations recently classified as belonging to a new species, T. aduncus, were each highly differentiated from populations of the truncatus morphotype, and from each other, suggesting a possible third species represented by the South African aduncus type.

Animals↗