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At least 505 records · Page 28Linked to original sources
PubMed1997

Sources of variability in data from a positive selection lacZ transgenic mouse mutation assay: an interlaboratory study.

Experimental features of a positive selection transgenic mouse mutation assay based on a lambda lacZ transgene are considered in detail, with emphasis on results using germ cells as the target tissue. Sources of variability in the experimental protocol that can affect the statistical nature of the observations are examined, with the goal of identifying sources of excess variation in the observed mutant frequencies. The sources include plate-to-plate (within packages), package-to-package (within animals), and animal-to-animal variability. Data from five laboratories are evaluated in detail. Results suggest only scattered patterns of excess variability below the animal-to-animal level, but, generally, significant excess variability at the animal-to-animal level. Using source of variability analyses to guide the choice of statistical methods, control-vs-treatment comparisons are performed for assessing the male germ cell mutagenicity of ethylnitrosourea (ENU), isopropyl methanesulfonate (iPMS), and methyl methanesulfonate (MMS). Results on male germ cell mutagenesis of ethyl methanesulfonate (EMS) and methylnitrosourea (MNU) are also reported.

Animals↗
PubMed1994

Bcl-2 is upregulated at the CD4+ CD8+ stage during positive selection and promotes thymocyte differentiation at several control points.

In vivo thymocyte maturation models were used to investigate the differentiation role of Bcl-2. In alpha/beta T cell receptor (TCR) class II-restricted transgenic mice, Bcl-2 was upregulated at the CD4+ CD8+ stage during positive selection. The lckpr-bcl2 transgene was bred onto MHC classes I-I- and II-I-, MHC-I-, and alpha/beta TCR backgrounds to determine whether Bcl-2 promoted thymocyte maturation in the absence of coreceptor-MHC interaction. Bcl-2 rescued CD8+ thymocytes in class I-I- and alpha/beta TCR in mice; however, they were not exported to the periphery. Bcl-2 had no effect on CD4 lineage maturation in class II-I- mice. No single-positive thymocytes accumulate in MHC-I- mice despite overexpressed Bcl-2. Thus, Bcl-2 enables selection of certain TCRs on class II molecules and their differentiation along the CD8 pathway; however, Bcl-2 did not substitute for positive selection. In RAG-1-I- mice, Bcl-2 promoted differentiation to the CD4+ CD8+ stage. Bcl-2 can promote thymocyte maturation at several control points.

Animals↗
PubMed2000

Affiliation to mature B cell repertoire and positive selection can be separated in two distinct processes.

Using an 'oligoclonal' model, we have previously shown that mice transgenic for a mu chain (H3) and deficient for kappa chain expression display a mature B cell repertoire largely dominated by the H3/lambda1 pair, while the four H3/lambda available combinations can be observed in the immature B cell compartment. This led us to propose the existence of a positive selection process. To test this hypothesis, we have introduced the SJL lambda locus coding for a defective lambda1 chain (lambda1(s)) that creates a dysfunctional Ig receptor complex during B cell differentiation. Our results show that the lambda1(s) defect impairs the development of mature B cells when the H3-mu transgene insert is present in the hemizygous state. This suggests that the Gly --> Val substitution present in the C(lambda)1(s) chain at position 155 is sufficient to abrogate the selection of the H3/lambda1 pair. Unexpectedly, when the H3-mu transgene array is present in a homozygous state in lambda1(s) mice but not in 'wild-type' lambda1 mice (lambda1(+)), a significant number of mature B cells expressing all H3/lambda combinations can be developed. These results indicate that the overriding H3/lambda1 dominance observed in lambda1(+) mice is due to a positive selection process and not to a negative selection of other H3/lambda combinations. They also show that the export of B cells to the periphery can be controlled by the expression of the mu chain.

Amino Acid Substitution↗
PubMed1995

A novel positive selection for identifying cold-sensitive myosin II mutants in Dictyostelium.

We developed a positive selection for myosin heavy chain mutants in Dictyostelium. This selection is based on the fact that brief exposure to azide causes wild-type cells to release from the substrate, whereas myosin null cells remain adherent. This procedure assays myosin function on a time scale of minutes and has therefore allowed us to select rapid-onset cold-sensitive mutants after random chemical mutagenesis of Dictyostelium cells. We developed a rapid technique for determining which mutations lie in sequences of the myosin gene that encode the head (motor) domain and localized 27 of 34 mutants to this domain. We recovered the appropriate sequences from five of the mutants and demonstrated that they retain their cold-sensitive properties when expressed from extrachromosomal plasmids.

Animals↗
PubMed1993

CD8 is needed for positive selection but differentially required for negative selection of T cells during thymic ontogeny.

During thymic development, immature thymocytes expressing major histocompatibility complex (MHC) class I-restricted T cell receptors (TcR) differentiate into CD8+ T cells with cytolytic functions. To evaluate the role of CD8 in positive and negative selection during thymic ontogeny, mice rendered CD8-null by gene targeting were bred with three lines of transgenic mice expressing unique MHC class I-restricted TcR. In all three instances CD8 was required for positive selection of MHC class I-restricted transgenic T cells. The efficiency of positive selection decreased in accordance with a reduced level of CD8 expression on thymocytes. Surprisingly, there was a differential requirement for CD8 expression in negative selection of MHC class I-restricted thymocytes, depending on the antigen specificity of TcR. These observations show that CD8 is essential for positive selection but is differentially required for negative selection of MHC class I-restricted T cells. Thus thymic selection, at least for negative selection, can occur in the absence of the CD8 accessory molecule.

Animals↗
PubMed1998

Rapid deletion of rearranged T cell antigen receptor (TCR) Valpha-Jalpha segment by secondary rearrangement in the thymus: role of continuous rearrangement of TCR alpha chain gene and positive selection in the T cell repertoire formation.

A rearranged T cell receptor (TCR) Valpha and Jalpha gene from a cytochrome c-specific T cell hybridoma was introduced into the genomic Jalpha region. The introduced TCR alpha chain gene is expressed in a majority of CD3 positive and CD4 CD8 double-negative immature thymocytes. However, only a few percent of the double-positive and single-positive thymocytes express this TCR alpha chain. This decrease is caused by a rearrangement of TCR alpha chain locus, which deletes the introduced TCR gene. Analysis of the mice carrying the introduced TCR alpha chain and the transgenic TCR beta chain from the original cytochrome c-specific T cell hybridoma revealed that positive selection efficiently rescues double-positive thymocytes from the loss of the introduced TCR alpha chain gene. In the mice with negatively selecting conditions, T cells expressing the introduced TCR alphabeta chains were deleted at the double-positive stage. However, a large number of thymocytes escape negative selection by using an endogenous TCR alpha chain created by secondary rearrangement maintaining normal thymocyte development. These results suggest that secondary rearrangements of the TCR alpha chain gene play an important role in the formation of the T cell repertoire.

Animals↗
PubMed1994

Functional commitment to helper T cell lineage precedes positive selection and is independent of T cell receptor MHC specificity.

Thymocyte differentiation proceeds from double positive CD4+CD8+ to single positive T cells. It has been proposed that this process occurs by an instructive or a stochastic mechanism. In this report, we show that in recombination-deficient mice (RAG-1-I-) constitutive expression of a CD8 transgene allows maturation of CD4+(CD8tg+) cells, which express mature levels of a transgenic class I-restricted T cell receptor, F5. Rescued F5+CD4+(CD8tg+) cells have equivalent levels of T cell receptor expression as CD8end+ cells, respond to cognate antigen and, upon stimulation, they exhibit a phenotype characteristic of CD4+ helper T cells. These data are consistent with a model of differentiation that predicts that thymocytes become functionally committed to a helper or cytotoxic lineage before the final step of positive selection and independently of MHC specificity of their T cell receptor.

Animals↗
PubMed1980

A plasmid cloning vehicle allowing a positive selection for inserted fragments.

We describe a plasmid cloning vehicle, pTR262, which allows a strong positive selection (resistance to tetracycline) for transformants bearing plasmids which have DNA insertions. pTR262 is derived from plasmid pBR322 and contains the cI gene and adjacent regulator region oRpR or the bacteriophage lambda. The expression of the tetracycline resistance (tet-r) gene(s) in pTR262 requires transcription from pR and is repressed by the cI gene product, lambda repressor. Insertion of a DNA fragment into the HindIII or Bc/I sites in pTR262 inactivates the cI gene and allows expression of the tet-r gene(s) in the host bacterium. A 100-fold increase in the number of tetracycline-resistant transformants is obtained when HindIII- or Bc/I-generated fragments are added to a ligation mixture containing HindIII- or Bc/I-digested pTR 262 DNA.

DNA Restriction Enzymes↗
PubMed2006

Construction of a positive selection marker by a lethal gene with the amber stop codon(s) regulator.

A novel positive selection marker for Escherichia coli transformation was developed. The marker consisted of a DNA fragment encoding the C-terminal ribonuclease domain (CRD) of colicin E3 (colE3) and one or more amber stop codons between the initiation codon and the E3-CRD coding sequence. The toxicity of the marker was controlled by the suppressor activity the host cells possessed. This allowed both effective selection and propagation of the vector possessing the maker by selecting appropriate hosts from among those widely distributed: sup+ strains for selection and sup0 strains for propagation respectively. The insert DNA fragment was introduced onto the vector by replacing the marker DNA. The transformants harboring the vector with an insert grew, but those without an insert were effectively removed by the killing activity of E3-CRD encoded on the marker DNA. The marker was also successfully applied to lambda phage display vector.

Codon, Terminator↗
PubMed1990

Functional properties of CD19+ B lymphocytes positively selected from buffy coats by immunomagnetic separation.

Here we report that human B lymphocytes can be positively selected directly from buffy coats applying the anti-CD19 antibody AB1 coupled to magnetic beads. This isolation protocol is highly efficient and the isolated cell population is of very high purity and viability. As judged by cell cycle analysis and various parameters for cell activation, the cells are still in a resting state after isolation. Furthermore, different functional assays have shown that the isolation procedure does not interfere with either activation or proliferation/differentiation of CD19 selected cells as compared to negatively isolated cells. As a consequence of cross-linking during the isolation process, the CD19 antigen is temporarily down-regulated as measured by AB1 binding. Despite this decreased expression, monoclonal antibodies to the CD19 antigen nevertheless inhibited anti-mu plus B cell growth factor induced B cell activation as reported also for negatively isolated cells. Taken together, the presented data strongly suggest that B cells isolated through the CD19 antigen can be used in critical functional assays.

Antibodies, Anti-Idiotypic↗
PubMed1996

A positive selection vector for the analysis of structural plasmid instability in Bacillus subtilis.

A system for the positive selection of structural plasmid rearrangements in Bacillus subtilis was developed. Random deletions removing a transcription terminator structure in the assay plasmid, designated pGP100, resulted in expression of the cat-86 gene, under control of a constitutive bacteriophage promoter. The resulting chlorampenicol-resistant colonies were analyzed for plasmid contents and were shown, by restriction analysis, to contain initially both the intact parental plasmid and a deletion variant. Sequence analysis of deletion derivatives revealed a consensus target site (5'-A-T-T-A-A/T-3') at or near deletion termini, which resembles topoisomerase I target sites. Endpoints on one side of the deletions were found to be clustered in the promoter region of the tetracycline resistance gene present on pGP100, the gene product of which is an integral membrane protein. Furthermore, deletion of the genes encoding the ATP-dependent exonuclease, AddAB, severely reduced the structural stability of pGP100. The data indicate that similar mechanisms underlie deletion formation in pGP100, and a different plasmid-based system, pGP1, which we have analyzed previously.

Bacillus subtilis↗
PubMed1986

Characterization of human mononuclear cells after positive selection with immunomagnetic particles.

We have investigated the possibility to employing magnetic monodisperse polymer particles for positive selection of human peripheral blood mononuclear cell populations. By carefully titrating the ratio between particles and cells we succeeded in isolating a number of cell populations that could be cultivated subsequently in vitro for functional studies. The success of the procedure is partly dependent on the properties of the monoclonal antibodies used to sensitize the cells. Provided these antibodies do not react with membrane structures involved in the transduction of activating signals, highly purified, quiescent cell populations can be recovered in a single fractionation step. In most instances particles will detach from the isolated cells by overnight culture, and the particles can then be removed from the system by a suitable magnet. T lymphocytes, subpopulations of T lymphocytes, and B lymphocytes have been isolated in this way and studied in a variety of functional assay systems. Comparison with cells obtained after negative selection clearly demonstrates the usefulness of this technique, especially if the membrane marker selected for it is not directly engaged in the activation processes.

Antibodies, Anti-Idiotypic↗
PubMed1991

Circumsporozoite protein genes of malaria parasites (Plasmodium spp.): evidence for positive selection on immunogenic regions.

The circumsporozoite (CS) protein is a cell surface protein of the sporozoite, the stage of the life cycle of malaria parasites (Plasmodium spp.) that infects the vertebrate host. Analysis of DNA sequences supports the hypothesis that in Plasmodium falciparum, positive Darwinian selection favors diversity in the T-cell epitopes (peptides presented to T cells by host MHC molecules) of the CS protein. In gene regions encoding T cell epitopes of P. falciparum, the rate of nonsynonymous nucleotide substitution is significantly higher than that of synonymous substitution, whereas this is not true of other gene regions. Furthermore nonsynonymous nucleotide substitutions in these regions cause a change of amino acid residue charge significantly more frequently than expected by chance. By contrast, in Plasmodium cynomolgi, the same regions show no evidence of positive selection, and residue charge is conserved. The CS protein has a central repeat region, which is the target of host antibodies. In P. falciparum, the amino acid sequence of the repeat region is conserved within and between alleles. In P. cynomolgi, on the other hand, there is evidence that positive selection has favored evolution of two different repeat types within a given allele.

Amino Acid Sequence↗
PubMed2005

Directional positive selection on an allele of arbitrary dominance.

Most models of positive directional selection assume codominance of the beneficial allele. We examine the importance of this assumption by implementing a coalescent model of positive directional selection with arbitrary dominance. We find that, for a given mean fixation time, a beneficial allele has a much weaker effect on diversity at linked neutral sites when the allele is recessive.

Algorithms↗
PubMed2004

Positive selection detection in 40,000 human immunodeficiency virus (HIV) type 1 sequences automatically identifies drug resistance and positive fitness mutations in HIV protease and reverse transcriptase.

Drug resistance is a major problem in the treatment of AIDS, due to the very high mutation rate of human immunodeficiency virus (HIV) and subsequent rapid development of resistance to new drugs. Identification of mutations associated with drug resistance is critical for both individualized treatment selection and new drug design. We have performed an automated mutation analysis of HIV Type 1 (HIV-1) protease and reverse transcriptase (RT) from approximately 40,000 AIDS patient plasma samples sequenced by Specialty Laboratories Inc. from 1999 to mid-2002. This data set provides a nearly complete mutagenesis of HIV protease and enables the calculation of statistically significant K(a)/K(s) values for each individual amino acid mutation in protease and RT. Positive selection (i.e., a K(a)/K(s) ratio of >1, indicating increased reproductive fitness) detected 19 of 23 known drug-resistant mutation positions in protease and 20 of 34 such positions in RT. We also discovered 163 new amino acid mutations in HIV protease and RT that are strong candidates for drug resistance or fitness. Our results match available independent data on protease mutations associated with specific drug treatments and mutations with positive reproductive fitness, with high statistical significance (the P values for the observed matches to occur by random chance are 10(-5.2) and 10(-16.6), respectively). Our mutation analysis provides a valuable resource for AIDS research and will be available to academic researchers upon publication at http://www.bioinformatics.ucla.edu/HIV. Our data indicate that positive selection mapping is an analysis that can yield powerful insights from high-throughput sequencing of rapidly mutating pathogens.

Amino Acid Sequence↗
PubMed1981

D-Amino acid dehydrogenase of Escherichia coli K12: positive selection of mutants defective in enzyme activity and localization of the structural gene.

A method for the positive selection of dadA mutants defective in D-amino acid dehydrogenase has been devised. It consists in isolating mutants resistant to beta-chloro-D-alanine and screening for mutant colony color on a special agar medium. All 70 Escherichia coli K12 dadA mutants isolated either by this method or by other selection procedures map at a locus which is near to hemA and closely linked with dadR. Since some of the dadA mutants are thermosensitive in D-methionine utilization in vivo and have thermolabile D-amino acid dehydrogenase in vitro, it is proposed that the dadA gene codes for the enzyme structure. The broad substrate specificity, apparent membrane localization, inducibility by alanine, and repressibility by glucose strongly suggest that the D-amino acid dehydrogenase coded by the dadA gene is a species variant of the enzyme described under the same name in Salmonella typhimurium. It may be identical or homologous with the enzymes described under the names alaninase, D-alanine oxidase or D-alanine dehydrogenase in E. coli K12 or B.

D-Amino-Acid Oxidase↗
PubMed1986

Positive-selection vectors utilizing lethality of the EcoRI endonuclease.

The construction and use of a series of positive-selection vectors are described. These plasmids encode EcoRI endonuclease, the synthesis of which is under the control of the lacUV5 promoter. The pKG2 plasmid encodes a wild-type EcoRI endonuclease. In the absence of EcoRI methylase, the endonuclease is lethal. Cloning into any of the unique restriction sites within the endonuclease-coding gene allows survival of the transformed EcoRI-methylase-less host. The pKGW and pKGS plasmids encode an altered EcoRI endonuclease which, when repressed in a lacIQ host, allows survival in the absence of the methylase. Induction with IPTG, however, results in cell death as a result of high-level EcoRI synthesis. Cloning into any of the unique restriction sites within the EcoRI gene of pKGW or pKGS allows survival of derepressed transformed cells. These vectors strongly select for cloning events which inactivate the endonuclease gene.

Chromosome Mapping↗