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PubMed2002

Gene flow among Calomys musculinus (Rodentia, Muridae) populations in Argentina.

Calomys musculinus is a Sigmodontinae rodent inhabiting periodically disturbed habitats in the central eastern plains of Argentina. It is the natural reservoir host of Junin virus, the etiological agent of Argentine Hemorrhagic Fever (AHF). In order to analyze the levels of gene flow among populations of this species, allozymic variability at 26 loci was studied in 291 individuals from the endemic zone of AHF and localities outside it. All populations showed high levels of polymorphism (He between 0.107 and 0.144; P95% between 38 and 54%). Individual loci f values were in most cases negative, although not significantly different from zero. Mean genetic differentiation among populations was low, but statistically significant (theta = 0.020; P < 0.01). There was no correlation between genetic and geographic distances between pairs of populations, and scatter of the pairwise points suggests that, at the regional scale, genetic drift is more influential than gene flow. This result can be interpreted as indicative of a relatively recent expansion of C. musculinus habitat and restricted on-going gene flow, which would be compatible with a relatively slow expansion rate of AHF.

Alleles↗
PubMed1995

Identification of malignant cells in multiple myeloma bone marrow with immunoglobulin VH gene probes by fluorescent in situ hybridization and flow cytometry.

Because it has been difficult to identify and separate malignant cells in human lymphoid malignancies, we have developed a flow cytometry-based fluorescent in situ hybridization (FISH) technique using immunoglobulin (Ig) heavy chain variable region (VH) gene probes. After obtaining the specific VH gene sequence expressed by the multiple myeloma IM-9 cell line and the malignant cells in five multiple myeloma patients, sense and antisense biotinylated single-stranded RNA probes were prepared by transcription from the malignant clone's VH DNA sequences. The cells from the IM-9 cell line and from the mononuclear bone marrow cells of multiple myeloma patients were fixed, hybridized with the above biotinylated RNA probes, incubated with streptavidin-phycoerythrin, and analyzed by FACS analysis. The myeloma cells stained positive with their own specific antisense VH biotinylated RNa probes, whereas sense and irrelevant antisense biotinylated probes demonstrated only background staining. Dilutional concentrations of the IM-9 cell line with normal bone marrow cells were also accurately quantitated by this procedure. The application of this technique will allow a more accurate assessment of tumor burden in patients with multiple myeloma and should permit an accurate method of tumor cell purification for clinical as well as biological studies. Furthermore, this technological advance should be equally effective at identifying specific VH gene-expressing cells in other lymphoid malignancies, as well as in nonmalignant B cell disorders.

Bacterial Proteins↗
PubMed1998

Mitochondrial control region polymorphism reveal high amount of gene flow in Fennoscandian willow tits (Parus montanus borealis).

We studied the genetic variability and differentiation of two Fennoscandian willow tit (Parus montanus borealis) populations located ca. 1000 km apart in Finland and Sweden by using the control region sequences of the mitochondrial DNA. Individual variation in the control region was extensive since all the 13 Finnish and the 12 Swedish individuals had unique haplotypes and the mean pairwise genetic distance resulted in 0.0052 (range 0.0008-0.0109). In the minimum spanning network connecting the genotypes, the two populations were completely intermingled. The mismatch distribution of the combined data set was very close to expected distribution of an expanding population. This result was supported by a significantly negative Tajima's D value. The sequence data indicate that (1) the long-term effective population size of the breeding willow tits has been large (122,000 and 110,000 females for the Finnish and the Swedish populations, respectively); and that (2) the gene exchange between distant localities is/has been extensive. Dispersal area for the Finnish females was estimated to be about 19,000-30,000 km2 and for the Swedish, 22,000-28,000 km2. Thus, the whole Fennoscandian population can be regarded as one panmictic unit, without any subdivisions to local demes. The amount of gene flow is remarkable because the willow tit has been considered a highly sedentary species. Provided that adult birds are site-tenacious, the gene flow must occur through juvenile summer dispersal, or irruptive autumn invasions, or both.

Animals↗
PubMed1996

Detection of CD8 T-cell expansions with restricted T-cell receptor V gene usage in infants vertically infected by HIV-1.

OBJECTIVE: To investigate the T-cell receptor (TCR) repertoire usage in infants born to mothers infected with HIV-1 in order to discern possible perturbations in TCR usage as a consequence of HIV-1 infection. DESIGN: Blood samples from five HIV-1-infected and six non-infected children born to HIV-1-seropositive mothers were collected at two to three timepoints during the first and second year of life and the TCR variable gene usage was determined. METHODS: Triple staining flow cytometry analysis using a panel of monoclonal antibodies (MAb) to TCR V alpha and V beta gene products and antibodies to CD4 and CD8 was performed. RESULTS: Frequent large expansions of CD8+ lymphocyte subpopulations bearing distinct V alpha and V beta gene products was seen in HIV-1-infected children (four out of five) but was rarely detected in uninfected children. CONCLUSION: The study demonstrated the frequent occurrence of persistent and clonal expansions of CD8+ T cells bearing distinct V alpha/V beta gene products in some HIV-1 vertically infected infants similar to those observed during primary infection in adults.

CD8-Positive T-Lymphocytes↗
PubMed1998

Gene expression in rod shaped cardiac myocytes, sorted by flow cytometry.

OBJECTIVE: Primary cardiac myocyte cultures are usually contaminated with variable parts of different cell types, such as fibroblasts, endothelial cells and smooth muscle cells. Thus, the objective of our study was to analyse the gene expression in a pure population. METHODS: To obtain an homogeneous population, cardiac myocytes from adult rats were fixed with ethanol and sorted by flow cytometry. This approach is suitable for isolating either single cells or up to several thousand cells. To measure the messenger ribonucleic acid (mRNA) expression of different genes at the level of a few rod-shaped myocytes, a cDNA library was created by polymerase chain reaction (PCR). RESULTS: Sorting by a fluorescence-activated cell sorter (FACS) resulted in pure rod-shaped cardiac myocytes and isolated RNA from these cells is undegraded, as shown by Northern blotting. We demonstrated both the expression of housekeeping genes, such as beta-actin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as well as the myocyte-specific transcripts, alpha-cardiac myosin heavy chain (alpha-MHC) and beta-MHC. Furthermore, we showed the induction of the immediately early gene c-fos at the level of ten sorted cells. CONCLUSIONS: This method allows one to study gene expression in different cell types within the heart, in tissue samples or to tackle the problem of heterogeneity within a cell population.

Animals↗
PubMed2001

Genetic diversity of Iberian populations of Bemisia tabaci (Hemiptera: Aleyrodidae) based on random amplified polymorphic DNA-polymerase chain reaction.

The genetic structure of six Iberian populations of the whitefly Bemisia tabaci, two of them biotype Q, one biotype B, and the other three a mixture of both, has been studied using random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR). A total of 336 individuals was completely discriminated by means of 234 scored bands. Separate analyses of molecular variance of haploid males and diploid females using the pairwise number of differences between haplotypes showed that biotypes contribute significantly more to the observed variability than populations within biotypes. On average, gene flow between two biotypes of the same population is lower than between populations of identical biotypes. On the basis of these results and the nondetection under natural conditions of a single hybrid, we consider that both biotypes are genetically isolated under the ecological conditions prevailing in the south Iberian Peninsula. All populations of biotype Q presented similar values of intrapopulational diversity, which were higher than the values shown by populations of biotype B.

Animals↗
PubMed2002

Postglacial dispersal of the European rabbit (Oryctolagus cuniculus) on the Iberian peninsula reconstructed from nested clade and mismatch analyses of mitochondrial DNA genetic variation.

Nested clade analysis was applied to cytochrome b restriction site data previously obtained on 20 natural populations of the European rabbit across the Iberian Peninsula to test the hypothesis of postglacial dispersal from two main refugia, one in the northeast and the other in the southwest. Apart from historical fragmentation that resulted in geographic discontinuity of two distinct mitochondrial DNA (mtDNA) clades A and B, patterns of haplotype genetic variability have been shaped mostly by restricted gene flow via isolation by distance. The distribution of tip versus interior haplotypes suggests that dispersal occurred from both the southwestern and northeastern groups. Dispersal from the southwest had a north and northwest direction, whereas from the northeast it had mostly a western and southern orientation, with subsequent overlap in a southeastern-northwestern axis across the Iberian Peninsula. The analysis of the pairwise mismatch distribution of a 179-181-bp fragment of the mtDNA control region, for seven of those populations, further supports the idea that major patterns of dispersal were in the direction of central Iberia. Additionally, rabbit populations do not show signs of any significant loss of genetic diversity in the recent past, implying that they maintained large population sizes and structure throughout the ice ages. This is congruent with the fact that the Iberian Peninsula was itself a glacial refugium during Quaternary ice ages. Nonetheless, climatic oscillations of this period, although certainly milder than in northern Europe, were sufficient to affect the range distributions of Iberian organisms.

Animals↗
PubMedDate not supplied

Extensive gene flow in human populations as revealed by protein and microsatellite DNA markers.

Population genetic studies are mainly based on the description of genetic variability and on interpopulational comparisons using genetic distance measures. The evolutionary dynamics of the populations are inferred from these parameters and accurate estimates of gene flow may be critical. The present study reevaluates the role of gene flow in human populations by different statistical methods from a number of microsatellite and protein polymorphism data. The estimated number of individuals exchanged per generation (Nm) was greater than 1 in all data sets with all statistical methods. The correlation between geographic and genetic distances suggests a pattern of isolation by distance, characteristic of demographic and genetic equilibrium conditions among populations worldwide. Thus the high values of Nm may be interpreted as a reflection of high gene flow between geographically close populations. As expected, gene flow appears to exert a pivotal role in the genetic history of humans.

Gene Frequency↗
PubMed2003

The phylogeography of dusky dolphins (Lagenorhynchus obscurus): a critical examination of network methods and rooting procedures.

We investigated the phylogeography and evolutionary history of dusky dolphins (Lagenorhynchus obscurus) using DNA sequences of the full mitochondrial cytochrome b gene in 124 individuals from the putative stocks off Peru, Argentina and Southwest Africa. While genetic differentiation within oceans is surprisingly low, there is no evidence for recent female gene flow between Atlantic and Pacific waters. Highest genetic variability in terms of sequence divergence and number of haplotypes is found in the Atlantic. Our analyses also indicate that the eastern South Pacific dusky dolphins stock should be considered a separate management unit. Given the high level of mortality experienced by the Peruvian dusky dolphin in local fishery activities, these findings have important implications for an objective management of the species. Furthermore, we analysed our mitochondrial sequence data with several widely used network estimation and rooting methods. The resulting intraspecific gene genealogies and rooting inferences exhibited substantial differences, underlying the limitations of some algorithms. Given that scientific hypotheses and management decisions depend strongly on inferred tree or network topologies, there is a clear need for a systematic comparative analysis of available methods. Finally, the present study indicates that (i) the dusky and the Pacific white-sided dolphins are sister species and (ii) not only the Westwind Drift hypothesis but also other models of dispersion are compatible with the current geographical distribution of dusky dolphins.

Animals↗
PubMed2000

Human immunoglobulin variable region gene analysis by single cell RT-PCR.

This protocol describes application of single cell reverse transcription polymerase chain reaction (RT-PCR) to the study of human immunoglobulin V region usage. The procedure begins with separation of peripheral blood mononuclear cells (PBMC) from human blood. The PBMC are stained with the B cell selective marker, anti-CD19. Stained B cells are sorted by flow cytometry and deposited, consecutively, one cell into each of an array of tubes. cDNA for one or more antibody variable regions (VH and/or VL) is synthesized with a primer (or primers) complementary to sequence(s) within the constant region (Cmu, Cgamma, Ckappa and/or Clambda). The cDNA is used as template for PCR amplification with gene or gene family specific primers. A second PCR is then performed with two nested primers to increase both the specificity and quantity of V region PCR products. The purified PCR products are sequenced directly and aligned to V region germline database and the Genbank database. Single cell RT-PCR is a fast and convenient way to analyze V region gene expression. It avoids the bias that may be introduced into V region cDNA library construction by the presence of highly variable levels of mRNA in different cells. The PCR products are obtained in quantities that can be cloned into bacterial expression vectors for production of recombinant V region protein domains.

Adult↗
PubMed2000

Population genetics of Chrysomela tremulae: a first step towards management of transgenic Bacillus thuringiensis poplars Populus tremula x .P. tremuloides.

Many strategies have been proposed for delaying the development of insect resistance to Bacillus thuringiensis (Bt). The current paradigm for Bt resistance management is the high dose-refuge strategy. For this strategy to be successful: (i) heterozygotes must be killed in treated areas, (ii) resistant alleles must be rare (frequency < 10-3), and (iii) there must be a high level of gene flow between populations to ensure random mating. We studied gene flow within and between populations with a view to managing the resistance of Chrysomela tremulae (Coleoptera: Chrysomelidae) to new transgenic, highly toxic poplars expressing a synthetic Bt gene. In this study, we assessed the extent of gene flow in C. tremulae within and between 16 sites in France and Belgium, using allozyme markers. We found a high level of genetic variability in C. tremulae, with a mean of 0.206 +/- 0.16. There were no obvious limitations to gene flow between populations of C. tremulae over large geographical distances (several hundreds of kilometres). Nevertheless, a very low level of genetic differentiation was observed between a site located in the south of France and the sampled sites from the Centre region.

Animals↗
PubMed2000

RAPD variation among and within small and large populations of the rare clonal plant Ranunculus reptans (Ranunculaceae).

In the pre-alpine region of Europe numbers and sizes of populations of the clonal lake shore plant Ranunculus reptans have declined because of the regulation of lake water levels. We investigated genetic variation among and within 17 populations of different size (cover 1-10 000 m) in R. reptans with RAPD (random amplified polymorphic DNA) profiles. We sampled 127 rosettes in 14 populations at Lake Constance and three populations at or near Lake Como. There was significant genetic variation between plants from the two lake regions (5.9%, analysis of molecular variance [AMOVA], P < 0.001), among populations within lake regions (20.4%, P < 0.001), and within populations (73.7%, P < 0.001). Under the assumptions of Wright's island model the variation among populations corresponds to a gene flow of N(e)m = 0.70. Within the 14 Lake Constance populations we detected significant genetic variation among subpopulations separated by only a few metres (4.0% of the within-population variation; P < 0.05). Molecular variance was 24% smaller in small populations covering <100 m area than in larger ones (P < 0.03), indicating that samples from large populations were genetically more variable than samples representing comparable areas of smaller populations. We conclude that gene flow among populations is very limited and that genetic drift has caused reduced genetic variability of smaller populations. Conservation of genetic variability in R. reptans requires persistence of large and also of small populations (because of population differentiation), and it could be enhanced by increasing the size of small populations (to counter genetic drift).

Journal Article↗
PubMed1992

Effects of demographic and ethnohistorical factors on average heterozygosities of South Amerindians.

Recent investigations have shown that average within-Tupi genetic distances differ from within-Carib distances, which is possibly due to differences in effective size of the populations belonging to these two linguistic stocks of South American tribes. The aim of this paper is to verify the influence of demographic factors and of interpopulation contacts on the degree of intragroup genetic variability of 18 South American native groups (eight Carib, seven Tupi, and three Gê). The mean per locus per individual heterozygosity (Hm) was studied for seven polymorphic systems and the distance from the centroid rii on Hm was evaluated with regression analysis according to Harpending and Ward's model. Data on intergroup contacts among the populations since the end of the eighteenth century were collected from an extensive review of the ethnohistorical literature. The level of Hm of the three linguistic stocks did not differ, which suggests a homogeneous within-group variation for these groups. In addition, there was no association between gene diversity and population size. On the other hand, demographic size correlated with rii, which suggest that gene frequencies in groups of larger populations more closely resemble the average gene frequencies of native South Americans. Values of rii differed between the stocks, and the average distance from the centroid of the Carib was about 2.6 times greater than that of the Tupi, in accordance with previous genetic distance analysis. It should also be emphasized that there was an important correlation between mean heterozygosity and the degree of historical intertribal contacts. This constitutes a particularly significant finding, suggesting the basic role of intertribal gene flow of the past two centuries on the level of present genetic variability of South American tribes.

Gene Frequency↗
PubMed1995

T cell development and repertoire of mice expressing a single T cell receptor alpha chain.

We examined T cell development and T cell repertoire in transgenic mice expressing a single T cell receptor (TCR) alpha chain derived from the H-2Db-lymphocytic choriomeningitis virus (LCMV)-specific cytolytic T lymphocyte (CTL) clone P14. To generate these alpha P14 mice, mice transgenic for the P14 TCR alpha chain were backcrossed to TCR alpha-deficient mice. Thymi from alpha P14 mice exhibited a marked decrease of mature CD4+8- and CD8+4- single-positive thymocytes comparable to thymi from TCR alpha-deficient mice. Correspondingly, the number of peripheral T cells was reduced in the CD4 (tenfold) and in the CD8 (twofold) subsets when compared to normal mice. T cells from alpha P14 mice generated a primary anti-LCMV CTL response when stimulated in vitro with LCMV in contrast to normal mice which require priming in vivo; elimination of LCMV in vivo was, however, not improved. Flow cytometric analysis of T cells with V beta-specific antibodies showed a diverse endogenous TCR V beta repertoire. Functional analysis of the T cell repertoire, however, revealed a strongly reduced (30-fold) allogeneic and the absence of a vesicular stomatitis virus-specific CTL response and an impaired ability to provide T cell help for antibody isotype switching. Thus, T cell selection in the thymus was impaired and the T cell repertoire was limited in mice expressing only one type of TCR alpha chain.

Animals↗
PubMed2002

Characterization of Herpesvirus saimiri-transformed T lymphocytes from common variable immunodeficiency patients.

Common variable immunodeficiency (CVID) is a very frequent but heterogeneous syndrome of antibody formation. The primary defect remains unknown, but many reports describe peripheral blood T lymphocyte dysfunctions in a substantial proportion of CVID patients, which may impair T--B cell collaboration. In order to investigate whether such putative defects were intrinsic to T cells or, rather, secondary to quantitative differences in T cell subset distribution, or to other described disorders, we have used Herpesvirus saimiri (HVS) for the targeted transformation of CVID CD4+ and CD8+ T cells and subsequent functional evaluation by flow cytometry of their capacity to generate cell surface (CD154, CD69) or soluble (IL-2, TNF-alpha, IFN-gamma) help after CD3 engagement. Unexpectedly, the results showed that 40 different CVID blood samples exposed to HVS gave rise with a significantly increased frequency to transformed CD4+ T cell lines, compared to 40 age-matched controls (27% versus 3%, P < or = 0.00002) suggesting the existence of a CVID-specific signalling difference which affects CD4+ cell transformation efficiency. The functional analysis of 10 CD4+ and 15 CD8+ pure transformed T cell lines from CVID patients did not reveal any statistically significant difference as compared to controls. However, half of the CD4+ transformed cell lines showed CD154 (but not CD69) induction (mean value of 46.8%) under the lower limit of the normal controls (mean value of 82.4%, P < or = 0.0001). Exactly the same five cell lines showed, in addition, a significantly low induction of IL-2 (P < or = 0.04), but not of TNF-alpha or IFN-gamma. None of these differences were observed in the remaining CD4+ cell lines or in any of the transformed CD8+ cell lines. We conclude that certain CVID patients show selective and intrinsic impairments for the generation of cell surface and soluble help by CD4+ T cells, which may be relevant for B lymphocyte function. The transformed T cell lines will be useful to establish the biochemical mechanisms responsible for the described impairments.

Adolescent↗
PubMed2000

Population structure and mating biology of the polygynous ponerine ant Gnamptogenys striatula in Brazil.

Gnamptogenys striatula is a polygynous ponerine ant, whose colonies contain either several differentiated queens or several gamergates. Population structure, queen mating frequency and deviation from random mating were investigated in a north-eastern Brazilian population. Eight workers from each of 33 queenright colonies and 17 queens and their progeny (20-40 offspring) were genotyped using eight variable microsatellite markers. Population differentiation tests indicated limited gene flow at the scale of several kilometres, and tests of isolation by distance revealed population viscosity at the scale of a few metres. This population structure, together with the frequent colony migrations and fissions observed in the field, suggest that new nests are founded by budding in G. striatula. Genetic data showed that 13 of our 17 queens were single-mated and four were double-mated. The estimation of the range of maximal frequency of double-mated queens in the population was 0.232-0.259, demonstrating that mating frequency is low in G. striatula. The low estimated mean relatedness between the 17 queens and their mates (-0. 04 +/- 0.49) indicated no evidence of inbreeding in G. striatula.

Animals↗
PubMed1996

Demographic genetics of brown trout (Salmo trutta) and estimation of effective population size from temporal change of allele frequencies.

We studied temporal allele frequency shifts over 15 years and estimated the genetically effective size of four natural populations of brown trout (Salmo trutta L.) on the basis of the variation at 14 polymorphic allozyme loci. The allele frequency differences between consecutive cohorts were significant in all four populations. There were no indications of natural selection, and we conclude that random genetic drift is the most likely cause of temporal allele frequency shifts at the loci examined. Effective population sizes were estimated from observed allele frequency shifts among cohorts, taking into consideration the demographic characteristics of each population. The estimated effective sizes of the four populations range from 52 to 480 individuals, and we conclude that the effective size of natural brown trout populations may differ considerably among lakes that are similar in size and other apparent characteristics. In spite of their different effective sizes all four populations have similar levels of genetic variation (average heterozygosity) indicating that excessive loss of genetic variability has been retarded, most likely because of gene flow among neighboring populations.

Alleles↗
PubMed2002

Population structure of Candida albicans, a member of the human flora, as determined by microsatellite loci.

This study examines the macrogeographic population structure of Candida albicans, a yeast commensal of humans, through a population genetic analysis of 5 microsatellite loci in 13 cities. The populations were predominantly clonal with some recombination. About 5% of the genetic variation is between populations and the overall pattern is one of intermediate differentiation. We did not find a single widespread genotype but instead found high, macrogeographic gene flow in these clinical populations; the most common genotype was limited to Atlanta and San Francisco. Homogeneity is evident within large geographic regions, such as Europe, Asia, and the USA, and isolation by distance accounted for 39% of the variation observed. Overall gene flow for a member of the human flora is variable but can be extensive, with an average of 4.5 migrants per generation (N(m)). Eastern hemisphere populations were less divergent than those of the Americas and Caribbean, consistent with the expansion of humans out of the eastern hemisphere.

Alleles↗