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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↗
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↗
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↗
PubMed2000

Relationships among cave swallow populations (Petrochelidon fulva) determined by comparisons of microsatellite and cytochrome b data.

The cave swallow (Petrochelidon fulva) is a polytypic species with isolated populations in northwestern South America, southwestern North America, Yucatan, Greater Antilles, and Florida. We compared microsatellite genotypes of at least five individuals each from five populations and cytochrome b sequence data of two individuals each from seven populations plus two outgroups. Microsatellite allelic diversity was substantial, and the North American populations were about equally variable. In contrast, the Ecuadorian population had far less genetic variation. Gene flow was apparent among populations, especially between Texas and Florida. Genetic structure was greater than in widespread North American species but less than that of sedentary Neotropical birds. Microsatellite genetic distances indicated a close affinity between Ecuadorian and northern populations, especially Texas and Florida, but this finding was inconsistent with cytochrome b data, which indicated that the Ecuadorian population is the clear outgroup to the northern populations. Its outgroup status is consistent with recent classifications that designate South American populations as their own species (P. rufocollaris). The cytochrome b data further suggested that the northern populations are divided into two clades: Texas/Yucatan and Florida/Greater Antilles. The microsatellite data incorrectly measured the diversity and affinities of Ecuadorian birds apparently because of an ascertainment bias that results from the use of heterologous PCR primers. Despite these problems in measuring phylogenetic relationships, the microsatellite data appeared to work well as a population genetic marker in indicating population structure and gene flow.

Alleles↗
PubMed2004

Temporal allozyme divergence in infrapopulations of the hemiurid fluke Lecithochirium fusiforme.

The effect of time on genetic differentiation was studied among infrapopulations of mature specimens of the hemiurid fluke, Lecithochirium fusiforme, a parasite of marine fishes. Genetic distances and genetic structure within and among different temporal samples of a geographical population were investigated using starch gel electrophoresis, by screening 6 polymorphic loci in 2 groups of infrapopulations corresponding to different sampling data, i.e., winter 1997-1998 and autumn 1998. The genetic distance among infrapopulations was low (D = 0.000-0.058 +/- 0.041). However, genetic divergence among infrapopulations from the same geographic location was clearly lower within each temporal sample (G(ST) = 0.021 and 0.034) than the corresponding value obtained for 12 infrapopulations sampled at different seasons of the year (G(ST) = 0.067). These results suggest the existence of a relatively important temporal effect that accounts for the differences in genetic variability among adult infrapopulations of L. fusiforme. Therefore, a hypothetical temporal gene flow favored by the existence of persistent life-cycle stages of this species in paratenic hosts is not sufficient to mask the temporal differentiation caused by genetic drift.

Animals↗
PubMed1996

Tracing the development of single memory-lineage B cells in a highly defined immune response.

To study the development of B lymphocyte memory, we identified and isolated splenic B cells expressing a highly defined antibody variable region that constitutes a reproducible and predominant component of the memory antibody response to p-azophenylarsonate (Ars). Isolation was achieved during the primary immune response by surface staining and flow cytometry using a specific anti-idiotypic antibody called E4, which recognizes this canonical V region, encoded by one set of V gene segments. The isolated E4+ cells displayed all of the phenotypic characteristics of germinal center centrocytes, including a low level of surface Ig, a lack of surface IgD, a high level of receptor for peanut agglutinin, and expression of mutated antibody V genes. E4+ B cells were first detected in the spleen 7-8 d after primary immunization, reached peak numbers from days 10-13, and waned by day 16. Surprisingly, at their peak, E4+ cells comprised only 40,000 of all splenocytes, and half of these failed to bind Ars. Using this number, we estimate the total number of Ars-specific memory-lineage cells in the spleen to be no more than 50,000 (0.1%) at any one time, and presumably far fewer that are committed to the memory pool. Chromosomal copies of rearranged V genes from single E4+ cells were amplified by nested PCR, and the amplified products were sequenced directly without cloning, using standardized conditions that disclose virtually no Taq polymerase errors. V gene sequence analyses of E4+ cells isolated from single mice confirmed their canonical nature and revealed that they were derived from few precursors. In the average mouse, the E4+ pool was derived from fewer than five canonical precursors. Somatic mutations were found within the V genes of almost all cell isolates. At day 13, a significant fraction of E4+ cells had mutations known to increase antibody affinity for Ars, suggesting they were products of at least one cycle of post-mutational antigen-driven selection. However, the lack of shared mutations by clonally related cells indicated that the selective expansion of mutant subclones typical of memory responses had not yet taken place. This was supported by the observation that half of the E4+ cells failed to bind Ars. Collectively, our results indicate that the memory compartment is a highly selected entity, even at relatively early stages of the primary immune response when somatic mutation and clonal selection are still in progress. If germinal centers are the source of memory B cells, our data suggest that B cell memory may be derived from only a small fraction of all germinal centers.

Amino Acid Sequence↗
PubMed2000

Paternity and gene dispersal in limber pine (Pinus flexilis James).

This study provides empirical information on intrapopulation gene dispersal via pollen, the size of genetic 'neighbourhoods', and interpopulation gene flow in a long-lived conifer, limber pine (Pinus flexilis). We used allozyme loci for a paternity analysis of 518 seeds produced in an isolated population of limber pine located in north-eastern Colorado, U.S.A., separated by 2 km from the nearest conspecific trees and nearly 100 km from populations in the Rocky Mountains. We also used indirect techniques (FST analyses) to estimate gene flow rates among subdivisions of the study population and among five widely separated populations. Within the main study population limber pine exhibited a polymorphism level of 50%, observed heterozygosity of 0.159, and 2.36 alleles per polymorphic locus. Mountain populations were slightly more variable. The main study population showed significant differentiation in allozymes among neighbouring subpopulations. The mean FST was 0.031 and the gene flow rate among subpopulations was estimated as 7.8 migrants per generation. Among widely separated populations the mean FST was 0.035 and the gene flow rate was estimated as 6.9 migrants per generation. The paternity analysis indicated a best estimate of 6.5% pollen immigration (minimum 1.1%) from populations 2 km to 100+ km away. For 4% of the seeds examined, paternity could be ascribed to a single tree in the study population. Fractional paternity and likelihood methods were used to estimate pollen dispersal distances for the remainder of the seeds. Mean pollen dispersal distance was estimated at 140 m using the fractional method, similar to results from the other techniques. This compares with a mean distance of 172 m between potential mates. These results suggest near-panmictic pollen dispersal over this population, which covers about 15 ha. The observed allozyme differences and surprisingly low estimates of among-subpopulation gene flow are ascribed to a probable restriction of gene dispersal by seeds.

Alleles↗