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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↗

Low genetic structure in an epiphytic Orchidaceae (Oncidium hookeri) in the Atlantic rainforest of South-eastern Brazil.

BACKGROUND AND AIMS: Oncidium hookeri is a neotropical species of epiphytic Orchidaceae found in the Brazilian Atlantic rainforest at the top of the Mantiqueira Range of mountains. The genetic variation of O. hookeri was studied to assess the distribution of genetic variability within and among six populations localized in Atlantic rainforest remnants. Gene flow among populations and the occurrence of recent bottlenecks were investigated in order to infer the degree of isolation of these populations. METHODS: Thirteen polymorphic loci were used for allozyme electrophoresis. The data were analysed by means of standard statistical approaches, to estimate gene diversity and the genetic structure of the populations. KEY RESULTS: The mean gene diversity and allelic richness were H(e) = 0.099 and A = 1.75, respectively. F-statistics revealed high heterozygote deficiencies in all populations (F(IS) = 0.43-0.82). Several rare alleles were found in all the populations, and three populations presented private alleles. Low genetic differentiation among O. hookeri populations was detected (F(ST) = 0.029); natural selection may be involved in PGM locus differentiation among populations. The genetic differentiation between paired populations was low, bearing no correlation with geographic distance (Mantel test: r = -0.34, P = 0.72). Only two populations showed signs of recent bottlenecks. CONCLUSIONS: The heterozygote deficiency found seems to be caused by pollinator behaviour; the low frequencies of several alleles of different loci can be maintained due to clonal propagation. Despite the stochastic nature of the wind-dispersal of seeds to long distances, this process may promote an effective gene flow among populations, thus avoiding genetic differentiation.

Brazil↗

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↗

[Genetic diversity of isoenzyme in Culex pipiens complex field populations sampling from distinct area of China].

Eight field populations of Culex pipiens complex collected from five provinces (Guangdong, Henan, Shandong, Beijing and Yunnan) in 2001 were used to study genetic diversity by starch gel electrophoresis. Data from seven loci (ME,MDH-1,MDH-2,MDH-3,GPD,EST-2,EST-3) of four isozymes were analyzed by software Biosys2.0 and FSTAT(Version 2.9.3). The results were as follows: (1) The values of He (from 0.098 to 0.41) indicated genetic variabilities of different degree in populations.(2)The low level of gene flow (Nm=0.64) could not prevent genetic drift to cause the gene differentiation between populations. The genetic diversity between populations attributed to the genetic diversity of total populations is small (Gst =0.303), and the great part is accumulated within populations (Hs/Dst=2). (3) The genetic structure of Culex pipiens complex population was the isolation-by-distance model. (4) The genetic identity (or genetic distance) revealed the scale of genetic differentiation between populations which related to the collection sites.

Alleles↗

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↗

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↗

Allozyme genotype--environment relationships in natural populations of Drosophila buzzatii.

Allozyme frequency data from natural populations of Drosophila buzzatii were analyzed for genotype--environment relationships. Allele frequency and heterozygosity at six loci polymorphic throughout eastern Australia and a number of environmental factors (both means and variabilities) were examined by a variety of multivariate techniques. Significant genotype--environment associations were found for five of the six loci, and after correcting for geographic location significant associations remained for Est-2 and Adh-1 gene frequencies and heterozygosities and for Pgm gene frequencies. The results are discussed in relation to selection and gene flow and provide the basis for laboratory studies to disentangle confounded effects of (1) environmental means and environmental variabilities and (2) allele frequency and heterozygosity, and thus to further test for and determine the nature of any natural selection at particular allozyme loci.

Alleles↗

Comparative analysis of flow cytometric techniques in assessment of ZAP-70 expression in relation to IgVH mutational status in chronic lymphocytic leukemia.

We compared 1 subjective and 5 objective flow cytometric methods to evaluate zeta-associated protein (ZAP-70) expression in relation to immunoglobulin heavy-chain variable-region (IgVH) gene mutational status in 154 samples from 125 patients with chronic lymphocytic leukemia (CLL). ZAP-70 expression determined by all methods used correlated with IgVH gene mutational status, but none of them demonstrated high concordance rates. Of the objective methods, ZAP-70 staining determined as a ratio of molecules of equivalent soluble fluorochrome intensity in CLL cells to that in normal B cells (ZAP-70+ staining in IgVH germline cases, 59%; ZAP-70- in IgVH mutated cases, 75%) or T cells (ZAP-70+ in IgVH germline cases, 66%; ZAP-70- in IgVH mutated cases, 57%) provides the best combination for assigning ZAP-70+ status to IgVH germline and ZAP-70- status to IgVH mutated cases. The subjective method based on ZAP-70 expression in natural killer/T cells gave a similar result, but reproducibility between laboratories may be difficult. Further studies on ZAP-70 expression in relation to clinical parameters may address whether ZAP-70 is an independent prognostic marker for CLL.

DNA Mutational Analysis↗

Host isolation and patterns of genetic variability in three populations of Teladorsagia from sheep.

We have used a mitochondrial marker to explore the population genetics of an economically important parasite of sheep, Teladorsagia. We examined diversity within and between parasites from three very different host populations, as well as within and between individual hosts. One of our study populations, the Soay sheep on Hirta, St Kilda, is unusually isolated with no sheep having been introduced to the island since 1932. Worm haplotypes from Hirta were compared with those from two other host populations. Remarkably, despite its historical isolation the Hirta population shows similar levels of within-population diversity to the other study populations. No divergence between the three Teladorsagia populations was found, consistent with gene flow between the populations. The high diversity within Teladorsagia populations provides compelling evidence that this variability is a general feature of parasitic nematode populations. Such diversity may be caused by high effective population size, coupled with an increased mutation rate for mtDNA, which has important implications for the spread of anthelmintic resistance in nematode populations.

Animals↗

A human immunoglobulin lambda locus is similarly well expressed in mice and humans.

Transgenic mice carrying a 380-kb region of the human immunoglobulin (Ig) lambda light (L) chain locus in germline configuration were created. The introduced translocus on a yeast artificial chromosome (YAC) accommodates the most proximal Iglambda variable region (V) gene cluster, including 15 Vlambda genes that contribute to >60% of lambda L chains in humans, all Jlambda-Clambda segments, and the 3' enhancer. HuIglambdaYAC mice were bred with animals in which mouse Igkappa production was silenced by gene targeting. In the kappa-/- background, human Iglambda was expressed by approximately 84% of splenic B cells. A striking result was that human Iglambda was also produced at high levels in mice with normal kappa locus. Analysis of bone marrow cells showed that human Iglambda and mouse Igkappa were expressed at similar levels throughout B cell development, suggesting that the Iglambda translocus and the endogenous kappa locus rearrange independently and with equal efficiency at the same developmental stage. This is further supported by the finding that in hybridomas expressing human Iglambda the endogenous L chain loci were in germline configuration. The presence of somatic hypermutation in the human Vlambda genes indicated that the Iglambda-expressing cells function normally. The finding that human lambda genes can be utilized with similar efficiency in mice and humans implies that L chain expression is critically dependent on the configuration of the locus.

Animals↗

Genetic variability in Hesperozygis ringens Benth. (Lamiaceae), an endangered aromatic and medicinal plant of southern Brazil.

Hesperozygis ringens Benth. (Lamiaceae) is an aromatic and medicinal plant, characterized by a high concentration of isopulegone; it is endemic to the mountains of southeast Rio Grande do Sul, Brazil, and currently is considered endangered. The present study evaluated the intra- and interpopulation genetic variability of H. ringens by means of RAPD and ISSR molecular markers. The results showed that H. ringens populations are genetically structured, with low gene flow between populations, confirming the fragmentation imposed by anthropic action. Populations from the two areas of occurrence are genetically different. Low intrapopulation variability and heterozygosity were detected, indicating genetic drift and inbreeding. Based on the data, conservation strategies are discussed.

Base Sequence↗

GM and KM allotypes in nine population samples of Sicily.

Serum samples of 864 unrelated healthy male and female individuals belonging to nine provinces of Sicily were types for Glm (1,2,3,17), G3m (5,10,11,13,14,15,16,21), and KM (1). With the exception of the Trapani sample these samples are characterized by the presence of five GM haplotypes: GM*1,17;21,26; GM*1,2,17;21,26; GM*1,3;5,10,11,13,14,26; GM*3;5,10,13,14,26; and GM*1,17;10,11,13,15,16. The interpopulation variability in the distribution of these haplotypes is considerable, which is especially due to haplotypes GM*1,3;5,10,11,13,14,26, and GM*3;5,10,13,14,26. However, no clear-cut clustering of the samples according to historical or geographical facts could be shown. Comparisons with other Italian populations reveal the considerable genetic difference of the Sicilians, which is in particular caused by the presence of the haplotypes GM*1,3;5,10,11,13,14,26, and GM*1,17;10,11,13,15,16. These haplotypes are quite uncommon in Europeans and may reflect gene flow from the Middle East. The KM phenotype and allele frequencies also show a considerable interpopulation variability among the nine Sicilian samples, but as for GM no distinction between eastern and western provinces of Sicily is present.

Adult↗

Immunofluorescent surface labelling, flow sorting and culturing of putative epidermal stem cells derived from small skin punch biopsies.

Basal keratinocytes of human epidermis strongly express the cell surface glycoprotein beta(1)-integrin, and putatively harbour epidermal stem cells. Selective sorting and culturing of keratinocyte stem cells forms the basis for studies on the role of these cells as targets for therapeutic intervention and gene therapy. Here we have studied variables which affect cell surface labelling for beta(1)-integrin, flow sorting and subsequent culturing of beta(1)-integrin-positive and beta(1)-integrin-negative keratinocytes. Keratinocytes were derived from small human skin punch biopsies (3 or 4 mm in diameter), and we tested a number of variables such as choice of proteolytic enzyme for cell isolation, cell concentration, fixation, storage of fixed cell suspensions and labelling conditions. In contrast to thermolysin treatment for cell isolation, trypsin treatment left most cell surface beta(1)-integrin molecules intact. Ethanol and paraformaldehyde fixation interfered with beta(1)-integrin detection, and unfixed cells gave the best results. Optimisation of all the individual steps resulted in a labelling protocol for reproducible staining and sorting of the cells. Sorted cells were seeded in 96-well plates (300 cells/well) and colonies were obtained in more than 50% of the wells with beta(1)-integrin-positive keratinocytes. In plates with beta(1)-integrin-negative cells, only 10% of the wells contained keratinocyte colonies. Flow sorted keratinocytes obtained by trypsin formed numerous colonies in cell culture experiments. In cell suspensions obtained with thermolysin, only sparse colonies were formed. We conclude that our methodology permits the use of small human tissue samples for cell labelling and sorting, while preserving the clonogenic potential.

3T3 Cells↗

Analysis of individual immunoglobulin lambda light chain genes amplified from single cells is inconsistent with variable region gene conversion in germinal-center B cell somatic mutation.

Responding B cells in specific immune responses diversify their immunoglobulin genes and are selected on their variant antigen receptors in the microenvironment of the germinal center. The patterns of mutations previously reported for immunglobulin (Ig) genes have supported mechanistic hypotheses of either error-prone DNA synthesis or templated variable region gene conversion as the underlying mechanism in the generation of these mutations. To assess the role of gene conversion in germinal-center somatic mutation, we chose to examine nucleotide changes in mouse lambda light chain genes which arose in response to a specific antigen. Laboratory mice possess three V lambda subexons, two of which differ from one another by only seven nucleotides, making these two subexons ideal for gene conversion. In the current study, we used six-parameter flow cytometry to isolate single lambda light chain-expressing germinal-center B cells from two different time points in a primary immune response. We then individually amplified and sequenced individual V lambda 1 genes from these single cells for mutational analysis. None of the 32 V lambda 1 genes, containing a total of 40 mutations, showed evidence of gene conversion from either of the other V lambda subexons. Features such as the replacement to silent ratio of the mutations documented at the earlier time point indicate an absence of antigen-driven selection. These data indicate that V region gene conversion does not contribute to germinal-center somatic mutation and that gene conversion is not responsible for targeting mutation specifically to rearranged Ig genes. The biological implications are discussed.

Amino Acid Sequence↗

Comparison of flow cytometric methods for the measurement of ZAP-70 expression in a routine diagnostic laboratory.

Chronic lymphocytic leukaemia (CLL) follows a variable clinical course with patient survival ranging from only a few years despite treatment, to several decades in patients who may never require clinical intervention. Determination of the mutational status of a patient's immunoglobulin heavy chain variable region (Ig V(H)) gene has been used to provide prognostic information, but this assay is not available in most laboratories. The discovery of the expression of the protein tyrosine kinase zeta-associated protein (ZAP)-70 in V(H)-unmutated CLL cases led to its proposal as a surrogate marker for V(H) status. This study investigated the measurement of ZAP-70 expression in CLL using different flow cytometric protocols. Two different antibodies and two different staining methods were compared. The Caltag ZAP-70 antibody and Fix & Perm kit were the easiest to use and were the most sensitive and specific combination, with 91% concordance between ZAP-70 expression and V(H) status. Three patients (9%) were discordant (two V(H) mutated/ZAP-70 positive, and one V(H) unmutated/ZAP-70 negative). No correlation existed between CD38 and either ZAP-70 expression or V(H) status. Measurement of ZAP-70 expression using the Caltag antibody/kit combination provides a standardized flow cytometric method that could be introduced into a routine CLL immunophenotyping panel in a clinical diagnostic laboratory.

Aged↗

Contamination of refuges by Bacillus thuringiensis toxin genes from transgenic maize.

Transgenic crops producing insecticidal toxins from Bacillus thuringiensis (Bt) are widely used to control pests, but their benefits will be lost if pests evolve resistance. The mandated high-dose/refuge strategy for delaying pest resistance requires planting refuges of toxin-free crops near Bt crops to promote survival of susceptible pests. We report that pollen-mediated gene flow up to 31 m from Bt maize caused low to moderate Bt toxin levels in kernels of non-Bt maize refuge plants. Immunoassays of non-Bt maize sampled from the field showed that the mean concentration of Bt toxin Cry1Ab in kernels and the percentage of kernels with Cry1Ab decreased with distance from Bt maize. The highest Bt toxin concentration in pooled kernels of non-Bt maize plants was 45% of the mean concentration in kernels from adjacent Bt maize plants. Most previous work on gene flow from transgenic crops has emphasized potential effects of transgene movement on wild relatives of crops, landraces, and organic plantings, whereas implications for pest resistance have been largely ignored. Variable Bt toxin production in seeds of refuge plants undermines the high-dose/refuge strategy and could accelerate pest resistance to Bt crops. Thus, guidelines should be revised to reduce gene flow between Bt crops and refuge plants.

Animals↗

Differences in levels of heterozygosity in populations of the common gudgeon (Gobio gobio, Cyprinidae) among adjacent drainages in Central Europe: an effect of postglacial range dynamics?

Twenty-nine allozyme loci analyzed in 295 common gudgeons (Gobio gobio) from the Rhine, the upper Danube and the Elbe river systems revealed variability measures of P = 0.590, H(e) = 0.066, and G(ST) = 0.1415. Gene flow was estimated at N(e)m = 1.88 over 223 river km in the Danube basin, and at N(e)m = 1.96 over 300 river km in the Rhine system. Isolation-by-distance was not observed. Danubian gudgeons proved significantly more heterozygous (H(e) = 0.106) than those from the Rhine (H(e) = 0.057) or the Elbe drainages (H(e) = 0.029). Nine polymorphic enzymes contributed to this difference, which probably indicates dispersal bottlenecking of the postglacial immigrants into the Atlantic drainages of the study area. Refugial bottlenecking of gudgeons from Atlantic drainages in a Pleistocene refuge located in oceanic northwest Europe also seems possible. Slightly deeper genetic lineages in the Danube (G(ST) = 0.0859) than in the Rhine (G(ST) = 0.0793) agree with this explanation, as does the greater mean genetic distance among pairs of population samples from the Danube (D = 0.0138) than from the Rhine (D = 0.0054). A genetic distance of D = 0.0085 separated Rhenish and Danubian gudgeons.

Animals↗

Biased T cell receptor V gene usage in tissues with periodontal disease.

In an attempt to characterize TCR V gene usage in human periodontally diseased tissue, V alpha 2, V beta 5.2-3, V beta 5.3, V beta 5.1, V beta 6.7, V beta 6.7, V beta 8 and V beta 12.1 expressions were examined. Serial cryostat sections obtained from 20 periodontitis and 9 gingivitis biopsies were then reacted with monoclonal antibodies directed to each repertoire. The technique was combined with a sensitive alkaline phosphatase-anti-alkaline phosphatase method. Peripheral blood was obtained from 10 periodontitis and 2 gingivitis patients. TCR repertoire was also quantified by flow cytofluorography with FITC-conjugated antibodies. Cells displaying binding of each antibody were counted. The proportions to CD3-positive cells were then calculated. The pattern of each TCR V gene product expression in inflamed gingiva exhibited individual variation, nevertheless, a consistent pattern emerged. The V beta 5 subfamily and V beta 6.7 were frequently used repertoires in gingiva, whereas the V alpha 2 and V beta 8 subfamily were underexpressed in most cases. Furthermore, the TCR V gene product expression in gingival tissue was biased compared with autologous peripheral blood. Three of 10 periodontitis subjects showed 1 or 2 strikingly overrepresented repertoire comparatively with autologous blood. In these 3 subjects V beta 6.7 was overexpressed in two cases and 5.2-3, V beta 8 and V beta 12.1 were overexpressed in one case. These results suggest that gingival T-cells are not randomly mobilized from peripheral blood and that local events influence the TCR repertoire at the level of T-cell recruitment or T-cell expansion.

Adult↗