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Multiple Clones (multiple + clone)

Distribution by Scientific Domains

Medical Sciences	50%
Life Sciences	50%

Selected Abstracts

Quantitative analysis of antennal mosaic generation in Drosophila melanogaster by the MARCM system

GENESIS: THE JOURNAL OF GENETICS AND DEVELOPMENT, Issue 6 2008
Carolina Gomez-Diaz
Abstract Mosaics have been used in Drosophila to study development and to generate mutant structures when a mutant allele is homozygous lethal. New approaches of directed somatic recombination based on FRT/FLP methods, have increased mosaicism rates but likewise multiple clones in the same individual appeared more frequently. Production of single clones could be essential for developmental studies; however, for cell-autonomous gene function studies only the presence of homozygous cells for the target recessive allele is relevant. Herein, we report the number and extension of antennal mosaics generated by the MARCM system at different ages. This information is directed to obtain the appropriated mosaic type for the intended application. By applying heat shock at 10 different developmental stages from 0,12 h to 6,7 days after egg laying, more than 50% of mosaics were obtained from 5,028 adults. Single recombinant clones appeared mainly at early stages while massive recombinant areas were observed with late treatments. genesis 46:283,288, 2008. © 2008 Wiley-Liss, Inc. [source]

A new strategy for studying In Vitro the drug susceptibility of clinical isolates of human hepatitis B virus

HEPATOLOGY, Issue 4 2004
David Durantel
Resistance of hepatitis B virus (HBV) to antivirals has become a major clinical problem. Our objective was to develop a new method for the cloning of naturally occurring HBV genomes and a phenotypic assay capable of assessing HBV drug susceptibility and DNA synthesis capacity in vitro. Viral DNA was extracted from sera and was amplified by polymerase chain reaction, and amplicons were cloned into vectors that enable, after cell transfection, the initiation of the intracellular HBV replication cycle. Single or multiple clones were used to transfect Huh7 cells. The viral DNA synthesis capacity and drug susceptibility were determined by measuring the level of intracellular DNA intermediate, synthesized in absence or presence of antiviral, using Southern blot analysis. We have developed, calibrated, then used this phenotypic assay to determine the drug susceptibility of HBV quasispecies isolated throughout the course of therapy from patients selected according to their mutation profile. A multiclonal and longitudinal analysis enabled us to measure the variation of drug susceptibility of different viral quasispecies by comparison of IC50/IC90s with standards. The presence of famciclovir- or lamivudine-induced mutations in the viral population caused a change in viral DNA synthesis capacity and drug susceptibility in vitro, demonstrating the clinical relevance of the assay. In conclusion, our phenotypic assay enables the in vitro characterization of DNA synthesis capacity and drug susceptibility of HBV quasispecies isolated from patients. This assay should allow a better monitoring of patients undergoing antiviral therapy, as well as the screening of novel drugs on emerging resistant strains. (Hepatology 2004;40:855,864). [source]

Novel classical MHC class I alleles identified in horses by sequencing clones of reverse transcription-PCR products

INTERNATIONAL JOURNAL OF IMMUNOGENETICS, Issue 6 2003
C. Chung
Summary Improved typing of horse classical MHC class I is required to more accurately define these molecules and to extend the number identified further than current serological assays. Defining classical MHC class I alleleic polymorphism is important in evaluating cytotoxic T lymphocyte (CTL) responses in horses. In this study, horse classical MHC class I genes were analyzed based on reverse transcription (RT)-PCR amplification of sequences encoding the polymorphic peptide binding region and the more conserved alpha 3, transmembrane and cytoplasmic regions followed by cloning and sequencing. Primer sets included a horse classical MHC class I-specific reverse primer and a forward primer conserved in all known horse MHC class I genes. Sequencing at least 25 clones containing MHC class I sequences from each of 13 horses identified 25 novel sequences and three others which had been described. Of these, nine alleles were identified from different horses or different RT-PCR and 19 putative alleles were identified in multiple clones from the same RT-PCR. The primer pairs did not amplify putative non-classical MHC class I genes as only classical MHC class I and related pseudogenes were found in 462 clones. This method also identified classical MHC class I alleles shared between horses by descent, and defined differences in alleles between horses varying in equine leukocyte antigen (ELA)-A haplotype as determined by serology. However, horses sharing ELA-A haplotypes defined by serotyping did not always share cDNA sequences, suggesting subhaplotypic variations within serologically defined ELA-A haplotypes. The 13 horses in this study had two to five classical MHC class I sequences, indicating that multiple loci code for these genes. Sequencing clones from RT-PCR with classical MHC class I-specific primers should be useful for selection of haplotype matched and mismatched horses for CTL studies, and provides sequence information needed to develop easier and more discriminating typing procedures. [source]

Genetic diversity and Wolbachia infection of the Drosophila parasitoid Leptopilina clavipes in western Europe

MOLECULAR ECOLOGY, Issue 5 2004
Bart A. Pannebakker
Abstract Wolbachia are maternally transmitted bacteria that alter their arthropod hosts' reproduction in various ways, including parthenogenesis induction (PI). Wolbachia -induced parthenogenesis can have drastic effects on the genetic structure of its host because it potentially reduces populations to clones without genetic exchange. However, Wolbachia -induced parthenogenesis does not inevitably result in a reduction of genetic variation of infected populations vs. uninfected populations, because the parthenogenetic populations are initially derived from uninfected populations and can thus show similar genetic variation. Here we investigate these issues in infected and uninfected populations of the Drosophila parasitoid Leptopilina clavipes in western Europe. Wasps from 19 sites in the Netherlands, France and northern Spain were screened for Wolbachia and analysed using amplified fragment length polymorphism (AFLP) markers. All the populations from the Netherlands and mid-France were infected with the same two strains of Wolbachia, whereas populations from the Pyrenees were not infected. The infected and uninfected populations show identical levels of genetic variation, but have clearly diverged genetically, indicating the presence of a barrier that prevents gene flow. Within the infected wasps two distinct genotypes were found at multiple localities, indicating the coexistence of multiple clones. The conditions promoting clonal coexistence in L. clavipes are discussed. [source]