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Results for “Positive selection”

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PubMed1994

Characteristics of an in vitro system of thymocyte positive selection.

We investigated the phenotypic changes accompanying maturation of CD4+CD8+ thymocytes in the presence of thymic stomal cells in vitro. Many of the features that are characteristic of positive selection in vivo, such as an ordered down-regulation of CD4 or CD8 together with up-regulation of the TCR-alpha beta complex, also occurred in vitro. Transient expression of CD69 was also observed, together with activation of IL-2 and IL-4 cytokine genes, providing further evidence that positive selection in vitro involves a pattern of gene regulation comparable to that seen in vivo.

Animals↗

PubMed1999

[Methods of mobilizing hematopoietic cells, their collection using cell separation, purging--processing of pathologic cells and enrichment with CD 34+ cells--negative and positive selection].

Autological transplantation of bone marrow as well as hematopoietic and precursor cells obtained from peripheral blood with the use of cell apheresis is a therapy applied in the treatment of hematological diseases and solid tumours. The mobilization of hematopoietic cells is performed by applying cytostatic drugs and/or recombinant growth factors (G-CSF, GM-CSF). The collection of CD 34+ cells is performed by using cell separators. An important role in the transplantation procedure is played by purging techniques of transplantation material from residual neoplastic cells (negative selection) or isolation of hematopoietic cells (positive selection). The considerable progress in this field is connected with the implementation of Immunoadsorbtive or magnetic methods as well as those of molecular biology. The improvement of the procedure of hematopoietic cells transplantation and its efficacy is caused by the stimulation of the hematopoiesis by using the combination of cytokines (G-CSF, GM-CSF, IL-3, SCF) and purging of the hematopoietic cells obtained from the cellular apheresis.

Antigens, CD34↗

PubMed2005

Enhanced synonymous site divergence in positively selected vertebrate antimicrobial peptide genes.

Nonrandom patterns associated with adaptively evolving genes can shed light on how selection and mutation produce rapid changes in sequences. I examine such patterns in two independent families of antimicrobial peptide genes: those in frogs, which are known to have evolved under positive selection, and those in flatfishes, which I show have also evolved under positive selection. I address two recently proposed hypotheses about the molecular evolution of antimicrobial peptide genes. The first is that the mature peptide region is replicated by an error-prone polymerase that increases the mutation rate and the transversion/transition ratio compared to the signal sequence of the same genes. The second is that mature peptides evolve in a coordinated fashion with their propieces, such that a change in net charge in one molecular region prompts an opposite change in charge in the other region. I test these hypotheses using alternative methods that minimize alignment errors, correct for phylogenetic nonindependence, reduce sequence saturation, and account for differing selection pressures on different regions of the gene. In both gene families I show that divergence at both synonymous and nonsynonymous sites within the mature peptide region is enhanced. However, in neither gene family is there evidence of an increased mutational transversion/transition ratio or coordinated evolution. My observations are consistent with either an elevated mutation rate in an adaptively evolving gene region or widespread selection on "silent" sites. These hypotheses challenge the assumption that mutations are random and can be measured by the synonymous substitution rate.

Amino Acid Sequence↗

PubMed2004

Peptide variants of viral CTL epitopes mediate positive selection and emigration of Ag-specific thymocytes in vivo.

During development, thymocytes carrying TCRs mediating low-affinity interactions with MHC-bound self-peptides are positively selected for export into the mature peripheral T lymphocyte pool. Thus, exogenous administration of certain altered peptide ligands (APL) with reduced TCR affinity relative to cognate Ags may provide a tool to elicit maturation of desired TCR specificities. To test this "thymic vaccination" concept, we designed APL of the viral CTL epitopes gp33-41 and vesicular stomatitis virus nucleoprotein octapeptide N52-59 relevant for the lymphocytic choriomeningitis virus-specific P14- and vesicular stomatitis virus-specific N15-TCRs, respectively, and examined their effects on thymocytes in vivo using irradiation chimeras. Injection of APL into irradiated congenic (Ly-5.1) mice, reconstituted with T cell progenitors from the bone marrow of P14 RAG2(-/-) (Ly-5.2) or N15 RAG2(-/-) (Ly-5.2) transgenic mice, resulted in positive selection of T cells expressing the relevant specificity. Moreover, the variants led to export of virus-specific T cells to lymph nodes, but without inducing T cell proliferation. These findings show that the mature T cell repertoire can be altered by in vivo peptide administration through manipulation of thymic selection.

Animals↗

PubMed2003

Positive selection on protein-length in the evolution of a primate sperm ion channel.

Positive Darwinian selection on advantageous point substitutions has been demonstrated in many genes. We here provide empirical evidence, for the first time, that positive selection can also act on insertion/deletion (indel) substitutions in the evolution of a protein. CATSPER1 is a voltage-gated calcium channel found exclusively in the plasma membrane of the mammalian sperm tail and it is essential for sperm motility. We determined the DNA sequences of the first exon of the CATSPER1 gene from 15 primates, which encodes the intracellular N terminus region of approximately equal to 400 aa. These sequences exhibit an excessively high frequency of indels. However, all indels have lengths that are multiples of 3 nt (3n indels) and do not disrupt the ORF. The number of indel substitutions per site per year in CATSPER1 is five to eight times the corresponding rates calculated from two large-scale primate genomic comparisons, which represent the neutral rate of indel substitutions. Moreover, CATSPER1 indels are considerably longer than neutral indels. These observations strongly suggest that positive selection has been promoting the fixation of indel mutations in CATSPER1 exon 1. It has been shown in certain ion channels that the length of the N terminus region affects the rate of channel inactivation. This finding suggests that the selection detected may be related to the regulation of the CATSPER1 channel, which can affect sperm motility, an important determinant in sperm competition.

Amino Acid Sequence↗

PubMed2004

Codon volatility as an indicator of positive selection: data from eukaryotic genome comparisons.

It has been suggested that codon volatility (the proportion of the point-mutation neighbors of a codon that encode different amino acids) can be used as an index of past positive selection. We compared codon volatility with patterns of synonymous and nonsynonymous nucleotide substitution in genome-wide comparisons of orthologous genes between three pairs of related genomes: (1) the protists Plasmodium falciparum and P. yoelii, (2) the fungi Saccharomyces cerevisiae and S. paradoxus, and (3) the mammals mouse and rat. Codon volatility was not consistently associated with an elevated rate of nonsynonymous substitution, as would be expected under positive selection. Rather, the most consistent and powerful correlate of elevated codon volatility was nucleotide content at the second codon position, as expected, given the nature of the genetic code.

Animals↗

PubMed1997

Targeted expression of major histocompatibility complex (MHC) class II molecules demonstrates that dendritic cells can induce negative but not positive selection of thymocytes in vivo.

It is well established that lymphoid dendritic cells (DC) play an important role in the immune system. Beside their role as potent inducers of primary T cell responses, DC seem to play a crucial part as major histocompatibility complex (MHC) class II+ "interdigitating cells" in the thymus during thymocyte development. Thymic DC have been implicated in tolerance induction and also by some authors in inducing major histocompatibility complex restriction of thymocytes. Most of our knowledge about thymic DC was obtained using highly invasive and manipulatory experimental protocols such as thymus reaggregation cultures, suspension cultures, thymus grafting, and bone marrow reconstitution experiments. The DC used in those studies had to go through extensive isolation procedures or were cultured with recombinant growth factors. Since the functions of DC after these in vitro manipulations have been reported to be not identical to those of DC in vivo, we intended to establish a system that would allow us to investigate DC function avoiding artificial interferences due to handling. Here we present a transgenic mouse model in which we targeted gene expression specifically to DC. Using the CD 11c promoter we expressed MHC class II I-E molecules specifically on DC of all tissues, but not on other cell types. We report that I-E expression on thymic DC is sufficient to negatively select I-E reactive CD4+ T cells, and to a less complete extent, CD8+ T cells. In contrast, it only DC expressed I-E in a class II-deficient background, positive selection of CD4+ T cells could not be observed. Thus negative, but not positive, selection events can be induced by DC in vivo.

Animals↗

PubMed1986

Positive selection of activated T cells of the T8 (CD8) sub-type by immunomagnetic separation.

By conjugating a monoclonal IgM antibody of CD8-specificity to magnetite-containing polymer particles, we have developed a rapid and simple one-step procedure for positive selection of T8 cells. The method ensured good yield and viability of pure T8 cells. The positively selected T8-fraction from MLC activated T cells retained their cytolytic capacity and gave a moderate proliferative response. Most of the proliferative response occurred in the negatively selected T4 population which showed only a marginal cytotoxic activity against PHA blasts as target cells. Transferrin receptor expression, as measured in a direct radiobinding assay, could also be demonstrated both among pure T4 and T8 cells upon MLC-activation.

Antigens, Differentiation, T-Lymphocyte↗

PubMed2001

Surface molecules essential for positive selection are retained but interfered in thymic epithelial cells after monolayer culture.

It has been reported that the three-dimensional structure of thymic epithelial cells (TECs) is responsible for thymic positive selection but that this ability disappears when TECs are cultured in monolayer. These results have supported the hypothesis that certain TEC-specific molecules are extinguished during monolayer culture. In this study, using MHC class II-restricted T-cell receptor transgenic mice, we demonstrated that preselected CD4(+)8(+) (DP) thymocytes were inhibited from developing into CD4(+)8(-) (CD4SP) cells in reaggregate thymus organ culture with monolayer-cultured TECs, but this inhibition was removed when TECs were cultured in monolayer with protein synthesis inhibitor or when the cultured TECs were treated with fixative. These results seem to be inconsistent with the previous hypothesis and indicate that monolayer culture allows TECs to retain the surface molecules necessary for positive selection but interferes with their function, which must be sustained for three dimensional structure.

Animals↗

PubMed2002

Detecting recent positive selection in the human genome from haplotype structure.

The ability to detect recent natural selection in the human population would have profound implications for the study of human history and for medicine. Here, we introduce a framework for detecting the genetic imprint of recent positive selection by analysing long-range haplotypes in human populations. We first identify haplotypes at a locus of interest (core haplotypes). We then assess the age of each core haplotype by the decay of its association to alleles at various distances from the locus, as measured by extended haplotype homozygosity (EHH). Core haplotypes that have unusually high EHH and a high population frequency indicate the presence of a mutation that rose to prominence in the human gene pool faster than expected under neutral evolution. We applied this approach to investigate selection at two genes carrying common variants implicated in resistance to malaria: G6PD and CD40 ligand. At both loci, the core haplotypes carrying the proposed protective mutation stand out and show significant evidence of selection. More generally, the method could be used to scan the entire genome for evidence of recent positive selection.

Africa↗

PubMed1984

Positive selection vectors: a small plasmid vector useful for the direct selection of Sau3A-generated overlapping DNA fragments.

A positive selection plasmid vector ( pLA7 ), containing a unique BclI site, and its use in the facile selection of a library of overlapping DNA fragments, generated by a partial digest with Sau3A, is described. Selection depends on the 5-fluorouracil + 5' AMP resistance of upp- ush - [ pLA7ush ::cloned DNA] cells, whereas upp- ush - [ pLA7ush +] cells are sensitive under the same conditions.

Chromosome Mapping↗

PubMed1984

Asparaginase II of Saccharomyces cerevisiae: positive selection of two mutations that prevent enzyme synthesis.

A positive selection method, D-aspartic acid beta-hydroxamate resistance, was used to isolate Saccharomyces cerevisiae strains lacking the ability to synthesize asparaginase II. Of 100 such mutant strains, 93 exhibited mutations which were allelic with asp3, a previously characterized mutation. The other seven strains carried a new mutation, asp6. The asp6 mutation segregated 2:2 in asp6 X wild-type crosses and assorted from the asp3 mutation in asp6 X asp3 crosses. All seven asp6 mutant isolates reverted at a relatively high frequency, whereas the asp3 mutant isolates did not revert under the same conditions. Various independent asp3 isolates were mated to give heteroallelic diploids, which when sporulated and spread on D-asparagine medium yielded no recombinant strains.

Asparaginase↗

PubMed2006

Role of positive selection in the retention of duplicate genes in mammalian genomes.

The question of how duplicate genes are retained in a population remains controversial. The duplication-degeneration-complementation model, which involves no positive selection, stipulates a higher retention rate of duplicate genes in a small population than in a large one. This model has been accepted by many evolutionists. However, we found considerably more retentions and fewer losses of duplicate genes in the mouse genome than in the human genome, although the population size of rodents is in general larger than that of primates. Indeed, in nearly every interval of synonymous divergence between duplicate genes, the number of gene retentions in mouse is larger than that in human. Our findings suggest a more important role of positive selection in duplicate retention than duplication-degeneration-complementation. In addition, certain functional categories show a higher tendency of lineage-specific expansion than expected, suggesting lineage-specific selection or functional bias in retained duplicates.

Animals↗

PubMed2002

The signature of positive selection at randomly chosen loci.

In Drosophila and humans, there are accumulating examples of loci with a significant excess of high-frequency-derived alleles or high levels of linkage disequilibrium, relative to a neutral model of a random-mating population of constant size. These are features expected after a recent selective sweep. Their prevalence suggests that positive directional selection may be widespread in both species. However, as I show here, these features do not persist long after the sweep ends: The high-frequency alleles drift to fixation and no longer contribute to polymorphism, while linkage disequilibrium is broken down by recombination. As a result, loci chosen without independent evidence of recent selection are not expected to exhibit either of these features, even if they have been affected by numerous sweeps in their genealogical history. How then can we explain the patterns in the data? One possibility is population structure, with unequal sampling from different subpopulations. Alternatively, positive selection may not operate as is commonly modeled. In particular, the rate of fixation of advantageous mutations may have increased in the recent past.

Adaptation, Biological↗

PubMed1997

Thymus-derived glucocorticoids regulate antigen-specific positive selection.

While it is generally believed that the avidity of the T cell antigen receptor (TCR) for self antigen/major histocompatibility complex (MHC) determines a thymocyte's fate, how the cell discriminates between a stimulus that causes positive selection (survival) and one that causes negative selection (death) is unknown. We have previously demonstrated that glucocorticoids are produced in the thymus, and that they antagonize deletion caused by TCR cross-linking. To examine the role of glucocorticoids during MHC-dependent selection, we examined thymocyte development in organ cultures in which corticosteroid biosynthesis was inhibited. Inhibition of glucocorticoid production in thymi from alpha/beta-TCR transgenic mice resulted in the antigen- and MHC-specific loss of thymocytes that normally recognize self antigen/MHC with sufficient avidity to result in positive selection. Furthermore, inhibition of glucocorticoid production caused an increase in apoptosis only in CD+CD8(+) thymocytes bearing transgenic TCRs that recognized self antigen/MHC. These results indicate that the balance of TCR and glucocorticoid receptor signaling influences the antigen-specific thymocyte development by allowing cells with low-to-moderate avidity for self antigen/MHC to survive.

Animals↗

PubMed1994

Thymic epithelial cell lines that mediate positive selection can also induce thymocyte clonal deletion.

Negative selection of potentially autoreactive thymocytes occurs mainly in the thymus and is thought to be induced primarily by interaction with bone marrow-derived cells. However, some studies have also reported a role for radioresistant thymic cells, which are probably epithelial in origin, in the deletion of thymocytes reacting to endogenous superantigens. We have previously demonstrated that thymic epithelial cell lines could induce thymocyte-positive selection in vivo. In this study, we assessed the potential of these cells to delete thymocytes reacting to the staphylococcal enterotoxin A or B superantigens in vitro. In the presence of staphylococcal enterotoxin A or B we found that all thymic epithelial cell lines used in this study were capable of activating T cell hybrids or deleting CD4+CD8+ thymocytes expressing an appropriate TCR. The extent of superantigen-mediated thymocyte deletion mediated by thymic epithelial cell lines was comparable to that mediated by a thymic macrophage cell line. Similar results were obtained with three phenotypically distinct thymic cell lines, suggesting that the ability to induce thymocyte deletion might be a general feature of various subsets of thymic epithelium. The observations provided in this study, combined with our previous demonstration that the same thymic epithelial cell lines can participate in positive selection, suggest that a given stromal cell population might be capable of taking part both in positive and negative selection of thymocytes.

Animals↗

PubMed1995

Isolation of Epstein-Barr virus-infected clones of the human T-cell line MT-2: use of recombinant viruses with a positive selection marker.

Certain classes of human T-cell lymphomas have been shown to be persistently infected with Epstein-Barr virus (EBV). To achieve an experimental system of persistent EBV infection in T cells, we used EBV recombinants with a positive selection marker. Infection of the human T-cell line MT-2 with EBV recombinants that had a hygromycin resistance gene and subsequent selection with this drug permitted isolation and long-term maintenance of EBV-infected MT-2 clones. For each clone, essentially 100% of cells were positive for EBV nuclear antigen. These MT-2 clones harbor monoclonal episomes of EBV DNA and stably express two EBV latent proteins, EBV nuclear antigen 1 and latent membrane protein 1. The growth of these EBV-infected MT-2 clones was slower than that of uninfected clones, suggesting that EBV affects regulation of T-cell proliferation. EBV recombinants with a positive selection marker will be a useful tool in establishing experimental systems of persistent EBV infection in certain non-B-cell lineages.

Cell Line↗

PubMed2000

Efficient production of transgenic cassava using negative and positive selection.

In order to improve the efficiency of cassava (Manihot esculenta Crantz) transformation, two different selection systems were assessed, a positive one based on the use of mannose as the selective agent, and a negative one based on hygromycin resistance encoded by an intron-containing hph gene. Transgenic plants selected on mannose or hygromycin were regenerated for the first time from embryogenic suspensions cocultivated with Agrobacterium. After the initial selection using mannose and hygromycin, 82.6% and 100% of the respective developing embryogenic callus lines were transgenic. A system allowing plant regeneration from only transgenic lines was designed by combining chemical selection with histochemical GUS assays. In total, 12 morphologically normal transgenic plant lines were produced, five using mannose and seven using hygromycin. The stable integration of the transgenes into the nuclear genome was verified using PCR and Southern analysis. RT-PCR and northern analyses confirmed the transgene expression in the regenerated plants. A rooting test on mannose containing medium was developed as an alternative to GUS assays in order to eliminate escapes from the positive selection system. Our results show that transgenic cassava plants can be obtained by using either antibiotic resistance genes that are not expressed in the micro-organisms or an antibiotic-free positive selection system.

Blotting, Southern↗