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Results for “Positive selection”

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PubMed2004

Genomic evidence for recent positive selection at the human MDR1 gene locus.

The MDR1 multidrug transporter regulates the traffic of drugs, peptides and xenobiotics into the body as well as sensitive tissues like the brain, germ cells and the developing fetus. Hence, it may influence an individual's response to drugs as well as his/her susceptibility to complex diseases in which environmental factors, especially xenobiotics, play a role. Polymorphisms within this gene, especially single-nucleotide polymorphism e26/3435(C/T), have been variously associated with differences in MDR1 expression, function, drug response and disease susceptibility. Here, we report the detailed characterization of the haplotype and linkage disequilibrium architecture of the entire 200 kb of the MDR1 gene in five world populations, namely, Chinese, Malays, Indians, Caucasians and African-Americans. We observed varied haplotype diversity across the entire gene in the different populations. The major haplotype mh5, which contains the subhaplotype e12/1236T-e21/2677T-e26/3435T, is highly represented among the four non-African populations, while mh7, which contains the subhaplotype e12/1236C-e21/2677G-e26/3435C, accounts for over a third of African-American chromosomes. These observations are inconsistent with a simple population evolution model, but instead are suggestive of recent historical events that have maintained such long range linkage disequilibrium. Using a modified long-range haplotype test, we found statistically significant evidence of recent positive selection for the e21/2677T and e26/3435T alleles in the Chinese population, and for the e26/3435T allele in the Malay population. Interestingly, we also detected evidence for positive selection of the alternative allele e26/3435C in the African-American population. These data suggest that independent mutational events may have occurred on the mh5 and mh7 haplotypes of the MDR1 gene to confer positive selection in the non-African and African-American populations, respectively.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2004

Positive selection of B cells expressing low densities of self-reactive BCRs.

B cell tolerance or autoimmunity is determined by selective events. Negative selection of self-reactive B cells is well documented and proven. In contrast, positive selection of conventional B cells is yet to be firmly established. Here, we demonstrate that developing self-reactive B cells are not always highly sensitive to the deletion mechanisms imposed by membrane-bound self-antigens. At low amounts, membrane-bound antigens allow survival of B cells bearing a single high affinity self-reactive B cell receptor (BCR). More importantly, we show that forced allelic inclusion modifies B cell fate; low quantities of self-antigen induce the selection and accumulation of increased numbers of self-reactive B cells with decreased expression of antigen-specific BCRs. By directly measuring antigen binding by intact B cells, we show that the low amounts of self-antigen select self-reactive B cells with a lower association constant. A fraction of these B cells is activated and secretes autoantibodies that form circulating immune complexes with self-antigen. These findings demonstrate that conventional B cells can undergo positive selection and that the fate of a self-reactive B cell depends on the quantity of self-antigen, the number of BCRs engaged, and on its overall antigen-binding avidity, rather than on the affinity of individual BCRs.

Animals↗

PubMed1992

Differentiation of an immature T cell line: a model of thymic positive selection.

Thymocyte differentiation is dependent upon recognition of major histocompatibility complex (MHC) molecules on thymic stroma, a process called positive selection. Here we describe an immature CD4+8+ T cell line derived from a TCR transgenic mouse that differentiates into CD4+8- cells in response to antigen and nonthymic antigen-presenting cells. When injected intrathymically, these cells differentiate in the absence of antigen. The ability of immature T cells to recognize MHC molecules in the absence of foreign antigen in the thymus can thus be attributed to a unique property of thymic antigen-presenting cells. These studies also demonstrate the phenotypic and functional changes associated with TCR-mediated T cell maturation and establish an in vitro model system of positive selection.

Animals↗

PubMed2000

[Successful hematologic reconstitution using CD34+ cells positively selected from cryopreserved autologous peripheral blood stem cells in a patient with malignant lymphoma].

Clinical use of CD34+ cells positively selected from cryopreserved peripheral blood stem cells (PBSC) has been limited, and there have been only a few reports of this procedure, mainly because clump formation decreases the proportion of CD34+ cells that can be recovered. A 49-year-old Japanese woman with non-Hodgkin's lymphoma (NHL) (follicular mixed, B cell, stage IVA) was treated with seven cycles of conventional chemotherapy and achieved partial remission. During hematopoietic recovery after the seventh course of chemotherapy, PBSC were harvested by continuous leukapheresis and cryopreserved. However, clonal rearrangement of the immunoglobulin heavy chain gene was detected in the PBSC by Southern blot analysis. After high-dose chemotherapy, CD34+ cells were positively immunoselected from the cryopreserved PBSC and infused into the patient at 1.97 x 10(6)/kg. The overall purity and recovery rate of the CD34+ cells were 72.2% and 65.0%, respectively. There were no severe adverse effects after PBSC transplantation, and the time required for recovery of neutrophils to over 0. 5 x 10(9)/l and platelets to over 50 x 10(9)/l was 11 and 21 days, respectively. Transplantation of CD34+ cells positively selected from cryopreserved PBSC provides engraftment ability similar to that of unmanipulated PBSC.

Antigens, CD34↗

PubMed2000

NF-kappa B is required for the positive selection of CD8+ thymocytes.

To examine the role of NF-kappaB in T cell development, we analyzed thymocyte ontogeny in transgenic (mutant I-kappaBalpha (mI-kappaBalpha)) mice that express a superinhibitory form of the NF-kappaB inhibitory protein, I-kappaBalpha (I-kappaBalpha(A32/36)), under the control of the T cell-specific CD2 promoter and enhancer. Thymi from mI-kappaBalpha mice contained increased numbers of double-positive (DP) and decreased numbers of both CD4(+) and CD8(+) single-positive cells, consistent with a block in DP thymocyte maturation. In addition, expression of CD69, a marker of positive selection, was decreased on DP thymocytes from the mI-kappaBalpha mice. To test directly whether NF-kappaB was required for positive or negative selection, we generated mI-kappaBalpha mice expressing the H-Y or 2C alphabeta TCR transgenes. Expression of the I-kappaBalpha(A32/36) transgene caused a block in the positive selection of CD8(+) single-positive cells in both strains of TCR transgenic animals. In contrast, negative selection was unaffected by expression of the I-kappaBalpha(A32/36) transgene. Taken together, these results identified a NF-kappaB-dependent transcriptional pathway that is selectively required for the positive selection of CD8(+) thymocytes.

Animals↗

PubMed1998

A positive selection vector for cloning of long polymerase chain reaction fragments based on a lethal mutant of the crp gene of Escherichia coli.

We have constructed a cloning vector with a tight positive selection for recombinant clones in Escherichia coli. The positive selection pressure results from a lethal mutation within the E. coli gene coding for the catabolite gene activator protein CAP, which is disrupted whenever a fragment is successfully inserted. Here, we show that this "suicide" vector, pCAPs, is suitable for cloning of PCR products as long as 9.3 kb into several unique restriction sites which are scattered throughout the lethal gene.

Cloning, Molecular↗

PubMed2000

New positive selection system based on the parD (kis/kid) system of the R1 plasmid.

The use of vectors that are designed to allow positive selection of recombinants facilitates cloning experiments in E. coli. Using kid, a lethal gene of the R1 plasmid parD locus, we generated pKID vectors leading to high selective efficiency of recombinants (greater than 90%). The E. coli bacterial host used to propagate these vectors produces the Kis protein, the natural antagonist of Kid. This new positive-selection system exhibits the same efficiency as the original ccdB-based selection vectors, pKIL (4). We also show that the ccdB and kid systems are independent. This property increases the potential of plasmidic poison-antidote systems for genetic applications and opens the door to a generation of new vectors containing the two selection systems.

Amino Acid Sequence↗

PubMed2003

Recognition of a specific self-peptide: self-MHC class II complex is critical for positive selection of thymocytes expressing the D10 TCR.

We examined the specificity of positive and negative selection by using transgenic mice carrying a variant of the D10 TCR. We demonstrate that a point mutation at position 51 within the CDR2alpha segment significantly reduces the avidity of this TCR for its cognate ligand, but does not impact recognition of nonself MHC class II molecules. Although structural studies have suggested that this TCR site interacts with the MHC class II beta-chain, the avidity of this TCR for its ligand and the function of the T cell can be reconstituted by a point mutation in the bound antigenic peptide. These data demonstrate that the bound peptide can indirectly alter TCR interactions by influencing MHC structure. Remarkably, reducing the avidity of this TCR for a specific antigenic peptide-MHC ligand has a dramatic impact on thymic selection. Positive selection of thymocytes expressing this TCR is nearly completely blocked, whereas negative selection on allogenic MHC class II molecules remains intact. Therefore, the recognition of self that promotes positive selection of the D10 TCR is highly peptide-specific.

Alleles↗

PubMed2006

CCR7-dependent cortex-to-medulla migration of positively selected thymocytes is essential for establishing central tolerance.

Immature CD4+CD8+ thymocytes, which are generated in the thymic cortex, are induced upon positive selection to differentiate into mature T lymphocytes and relocate to the thymic medulla. It was recently shown that a chemokine signal via CCR7 is essential for the cortex-to-medulla migration of positively selected thymocytes in the thymus. However, the role of the cortex-to-medulla migration in T cell development and selection has remained unclear. The present study shows that the developmental kinetics and the thymic export of mature thymocytes were undisturbed in adult mice lacking CCR7 or its ligands (CCR7L). The inhibition of sphingosine-1-phosphate-mediated lymphocyte egress from the thymus led to the accumulation of mature thymocytes in the cortex of CCR7- or CCR7L-deficient mice, unlike the accumulation in the medulla of normal mice, thereby suggesting that mature thymocytes may be exported directly from the cortex in the absence of CCR7 signals. However, the thymocytes that were generated in the absence of CCR7 or CCR7L were potent in causing autoimmune dacryoadenitis and sialadenitis in mice and were thus incapable of establishing central tolerance to organ-specific antigens. These results indicate that CCR7-mediated cortex-to-medulla migration of thymocytes is essential for establishing central tolerance rather than for supporting the maturation or export of thymocytes.

Animals↗

PubMed2003

Expression profiling of a transformed thymocyte cell line undergoing maturation in vitro identifies multiple genes involved in positive selection.

Biochemical and genetic studies of thymocyte maturation would be facilitated by the development of cultured cell lines that reflect stages of positive selection. We have derived a CD4(+)CD8(+)TCR(+) T-lymphoid cell line (M20) from a murine thymic tumor induced by a retrovirus carrying the v-myc oncogene (M-MuLV(myc)). M20 subclones undergo several aspects of positive selection in response to co-culture with a thymic stromal cell line (St3), including down-regulation of CD4 and CD8, and up-regulation of CD5 and TCR. M20 possesses a functional TCR complex, and ligation of this complex produces changes similar to co-culture with St3 stroma. Expression profiling of M20 cells in this system identified 23 genes previously shown to be important in thymocyte maturation, as well as several novel candidate genes. This system provides a new model to elucidate the molecular mechanisms of thymocyte maturation and TCR-mediated cell signaling in double-positive thymocytes.

Animals↗

PubMed1996

Evolution of hemopoietic ligands and their receptors. Influence of positive selection on correlated replacements throughout ligand and receptor proteins.

The rates of amino acid replacement in cytokines and their receptors are high and vary considerably. To determine whether this reflects the action of positive selection, rates of nonsynonymous DNA substitution were examined and found to exceed the synonymous substitution rate in certain exons of rodent IL-3, granulocyte-macrophage stimulating factor, and IL-4. To determine the extent to which positive selection could account for correlations between the amino acid replacement rates of hemopoietins and their receptors, rates were examined in various domains: the correlation with ligand rate was not confined to the ligand-binding domain of the receptor, but extended into the transmembrane and cytoplasmic domains and even to leader peptide domains of both ligand and receptor. As the majority of these replacements are unlikely to be strongly advantageous, different levels of both positive and purifying selection contribute to the extensive variation in hemopoietin/receptor evolutionary rates. Changes in a few residues critical for ligand-receptor interaction may be followed by changes of lesser selective importance in both molecules: replacements of growth hormone residues that form hydrogen or salt bridges with the receptor occur in lineages in which there are many concurrent replacements. A ligand/receptor rate correlation is not found between the seven-transmembrane receptors and their ligands, whose mature forms are often short and completely conserved. This study predicts that a minority of concurrent evolutionary changes in hemopoietins and their receptors reflect directly compensatory changes.

Amino Acids↗

PubMed1986

Use of the lysis gene of bacteriophage phi X174 for the construction of a positive selection vector.

DNA fragments generated by a variety of restriction enzymes can easily be cloned in the small (3.2-kb) positive-selection vector pUH84, which contains the modified lysis gene of bacteriophage phi X174 under transcriptional control of the lac promoter. Plasmid pUH84 does not yield transformants after introduction into Escherichia coli unless the lysis gene is inactivated by insertion of foreign DNA into one of the unique PstI, SalI, AccI, HincII, BamHI, or EcoRI sites. This highly efficient positive selection of recombinants requires neither the use of a distinct host strain nor a special induction of the lysis function. Transcription of fragments cloned into pUH84 may be effectively regulated by the lac promoter provided the host cells are cotransformed with the newly constructed plasmid pUH7 which carries the IQ allele of the lac repressor gene.

Bacteriophage phi X 174↗

PubMed1991

H-2K molecules positively select V beta 17a+ CD4(-)8+ T cells in bone marrow and thymic chimeras.

Population size of V beta 17a brightly positive cells among CD4(-)8+ thymocytes was analyzed in thymic chimeras as well as bone marrow (BM) chimeras in which SWR/J mice were used as BM donors and various strains of mice including H-2Kb mutant (bm) mice as recipients. It was shown that the proportion of V beta 17a+ CD4(-)8+ thymocytes was determined by H-2K molecules expressed on thymic epithelial cells. The highest proportion was observed in Ks and Kb thymuses, the intermediate proportion in Ks/q and Kk, and the lowest in Kq thymuses. Fine analysis of the H-2Kbm molecules involved in the positive selection revealed that the region important to the selection was located on the beta-pleated floor of antigen recognition site. According to the three-dimensional class I structure, this site appears not to be directly accessible to the T cell antigen receptor. Thus, the present finding suggests that the substitutions of amino acids at this site alter the shape and charge of the peptide binding site and eventually influence the positive selection of the V beta 17a+ T cell repertoire during differentiation.

Animals↗

PubMed2000

Development of a nonantibiotic dominant marker for positively selecting expression plasmids in multivalent Salmonella vaccines.

We report the novel application of a herbicide-resistance-based dominant marker for the positive selection of expression plasmids in Salmonella serovar vaccines. The beta-lactamase gene of the plasmid pTETnir15, which expresses fragment C of tetanus toxin (TetC), has been replaced with the bar gene marker. The new plasmid pBAT1 can be positively selected in vitro within Salmonella serovars in the presence of the herbicide DL-phosphinothricin. The expression of TetC remains unaltered, and the Salmonella enterica serovar Typhimurium vaccine strain is stable and immunogenic in vivo.

Acetyltransferases↗

PubMed1991

Negative and positive selection of antigen-specific cytotoxic T lymphocytes affected by the alpha 3 domain of MHC I molecules.

The alpha 1 and alpha 2 domains of major histocompatibility complex (MHC) class I molecules function in the binding and presentation of foreign peptides to the T-cell antigen receptor and control both negative and positive selection of the T-cell repertoire. Although the alpha 3 domain of class I is not involved in peptide binding, it does interact with the T-cell accessory molecule, CD8. CD8 is important in the selection of T cells as anti-CD8 antibody injected into perinatal mice interferes with this process. We previously used a hybrid class I molecule with the alpha 1/alpha 2 domains from Ld and the alpha 3 domain from Q7b and showed that this molecule binds an Ld-restricted peptide but does not interact with CD8-dependent cytotoxic T lymphocytes. Expression of this molecule in transgenic mice fails to negatively select a subpopulation of anti-Ld cytotoxic T lymphocytes. In addition, positive selection of virus-specific Ld-restricted cytotoxic T lymphocytes does not occur. We conclude that besides the alpha 1/alpha 2 domains of class I, the alpha 3 domain plays an important part in both positive and negative selection of antigen-specific cells.

Animals↗

PubMed2003

Use of borate to control the 5'-position-selective microbial glucosylation of pyridoxine.

Nearly 100% 5'-position selectivity of transglucosylation from maltodextrin to pyridoxine (PN) by cells of Verticillium dahliae TPU 4900 was observed when the reaction was carried out with borate. The same effect of borate was observed not only during synthesis of pyridoxine 5'-alpha-D-glucoside by partially purified enzyme of this strain but also during synthesis of this compound by other microorganisms and with other enzymes (alpha-glucosidase and cyclomaltodextrin glucanotransferase). The effect was thought to be caused by the formation of a borate complex with 3- and 4'-position hydroxyl groups of PN. A decrease in the formation of pyridoxine 5'-alpha-D-glucoside was observed in the reaction with borate, but this decrease was overcome by optimizing the pH and increasing the amount of cells in the reaction mixture.

Borates↗

PubMed2005

The signature of positive selection on standing genetic variation.

Considerable interest is focused on the use of polymorphism data to identify regions of the genome that underlie recent adaptations. These searches are guided by a simple model of positive selection, in which a mutation is favored as soon as it arises. This assumption may not be realistic, as environmental changes and range expansions may lead previously neutral or deleterious alleles to become beneficial. We examine what effect this mode of selection has on patterns of variation at linked neutral sites by implementing a new coalescent model of positive directional selection on standing variation. In this model, a neutral allele arises and drifts in the population, then at frequency f becomes beneficial, and eventually reaches fixation. Depending on the value of f, this scenario can lead to a large variance in allele frequency spectra and in levels of linkage disequilibrium at linked, neutral sites. In particular, for intermediate f, the beneficial substitution often leads to a loss of rare alleles--a pattern that differs markedly from the signature of directional selection currently relied on by researchers. These findings highlight the importance of an accurate characterization of the effects of positive selection, if we are to reliably identify recent adaptations from polymorphism data.

Biological Evolution↗

PubMed1998

Allogeneic peripheral stem cell transplantation using positively selected CD34+ cells from HLA-mismatched donors.

We examined five children who underwent allogeneic peripheral stem cell transplantation (PSCT) using positively selected CD34+ cells from three or two loci-mismatched donors. CD34+ cells mobilized from peripheral blood were separated by immunomagnetic beads. CD34+ cells at 2.2-6.2 x 10(6)/kg were transplanted into three patients with refractory leukemia, a patient with relapsed medulloblastoma and a patient with Fanconi's anemia following a conditioning regimen which included irradiation, alkylating agents and antithymocyte globulin treatment. The number of infused CD3+ cells included in grafts was 2.3-22.7 x 10(4)/kg. Four patients achieved engraftment and hematopoietic reconstitution (> 5 x 10(8)/l of neutrophils on day 10 or 11). Graft rejection was observed in the patient with Fanconi's anemia, but a rapid engraftment was obtained after second PSCT. Although no prophylactic agents other than ATG (included in the conditioning regimen) were used, greater than grade I acute GVHD was not observed, but limited chronic GVHD was observed in two patients. The two patients with leukemia relapsed on days 103 and 210, respectively, and the patient with medulloblastoma died of disease on day 159. The patient with Fanconi's anemia died of fungal infection. CMV and HHV-6 diseases developed in four and two patients, respectively. Thus, although SCT using positively selected peripheral CD34+ cells may be an alternative approach for overcoming graft rejection and GVHD from HLA- mismatched donors, persistent immune deficiency attributing to extremely low numbers of T cells in grafts can potentially lead to reactivation of herpes viruses.

Adolescent↗