A processed short transcript of Leishmania, ODD1.
Explore the source record for details and available documents.
Amino Acid Sequence↗
Biomedical subjects
L R Tosi
Publications and source records attributed to L R Tosi.
New Mos1 mariner transposons suitable for the recovery of gene fusions in vivo and in vitro.
The Drosophila Mos1 element can be mobilized in species ranging from prokaryotes to protozoans and vertebrates, and the purified transposase can be used for in vitro transposition assays. In this report we developed a 'mini-Mos1' element and describe a number of useful derivatives suitable for transposon mutagenesis in vivo or in vitro. Several of these allow the creation and/or selection of tripartite protein fusions to a green fluorescent protein-phleomycin resistance (GFP-PHLEO) reporter/selectable marker. Such X-GFP-PHLEO-X fusions have the advantage of retaining 5' and 3' regulatory information and N- and C-terminal protein targeting domains. A Mos1 derivative suitable for use in transposon-insertion mediated linker insertion (TIMLI) mutagenesis is described, and transposons bearing selectable markers suitable for use in the protozoan parasite Leishmania were made and tested. A novel 'negative selection' approach was developed which permits in vitro assays of transposons lacking bacterial selectable markers. Application of this assay to several Mos1 elements developed for use in insects suggests that the large mariner pM[cn] element used previously in vivo is poorly active in vitro, while the Mos1-Act-EGFP transposon is highly active.
Animals↗
Characterisation of three chromosomal ends of Leishmania major reveals transcriptional activity across arrays of reiterated and unique sequences.
The 36 chromosomes of the parasite Leishmania major range in size from 200 kb to approximately 2.5 Mb and variation between homologues seems to be restricted to the telomeric and subtelomeric regions. We have isolated three cosmids carrying the telomere hexameric repeat and assigned them to the extreme location of chromosomes 3, 7 and 20. When considering the distribution of repetitive sequences, Southern analysis of the three chromosomal ends indicated the existence of at least two classes of chromosomal extremities: one of them is composed almost exclusively of unique sequences and the other is characterised by patches of both reiterated and unique sequences. We devised a transfection-based strategy that allowed the determination of a map of transcripts in each of the regions examined. Sequencing of the chromosome 20 cosmid revealed the existence of a novel class of reiterated sequence, LST-R378, and 10 ORFs drawing a map of putative genes compatible with the map of transcripts.
Animals↗
cis and trans factors affecting Mos1 mariner evolution and transposition in vitro, and its potential for functional genomics.
Mos1 and other mariner / Tc1 transposons move horizon-tally during evolution, and when transplanted into heterologous species can transpose in organisms ranging from prokaryotes to protozoans and vertebrates. To further develop the Drosophila Mos1 mariner system as a genetic tool and to probe mechanisms affecting the regulation of transposition activity, we developed an in vitro system for Mos1 transposition using purified transposase and selectable Mos1 derivatives. Transposition frequencies of nearly 10(-3)/target DNA molecule were obtained, and insertions occurred at TA dinucleotides with little other sequence specificity. Mos1 elements containing only the 28 bp terminal inverted repeats were inactive in vitro, while elements containing a few additional internal bases were fully active, establishing the minimal cis -acting requirements for transposition. With increasing transposase the transposition frequency increased to a plateau value, in contrast to the predictions of the protein over-expression inhibition model and to that found recently with a reconstructed Himar1 transposase. This difference between the 'natural' Mos1 and 'reconstructed' Himar1 transposases suggests an evolutionary path for down-regulation of mariner transposition following its introduction into a naïve population. The establishment of the cis and trans requirements for optimal mariner transposition in vitro provides key data for the creation of vectors for in vitro mutagenesis, and will facilitate the development of in vivo systems for mariner transposition.
Animals↗
Purified mariner (Mos1) transposase catalyzes the integration of marked elements into the germ-line of the yellow fever mosquito, Aedes aegypti.
Derivatives of the mariner transposable element, Mos1, from Drosophila mauritiana, can integrate into the germ-line of the yellow fever mosquito, Aedes aegypti. Previously, the transposase required to mobilize Mos1 was provided in trans by a helper plasmid expressing the enzyme under the control of the D. psuedoobscura heat-shock protein 82 promoter. Here we tested whether purified recombinant Mos1 transposase could increase the recovery of Ae. aegypti transformants. Mos1 transposase was injected into white-eyed, kh(w)/kh(w), Ae. aegypti embryos with a Mos1 donor plasmid containing a copy of the wild-type allele of the D. melanogaster cinnabar gene. Transformed mosquitoes were recognized by partial restoration of eye color in the G(1) animals and confirmed by Southern analyses of genomic DNA. At Mos1 transposase concentrations approaching 100 nM, the rate of germ-line transformants arising from independent insertions in G(0) animals was elevated 2-fold compared to that seen in experiments with helper plasmids. Furthermore, the recovery of total G(1) transformants was increased 7.5-fold over the frequency seen with co-injected helper plasmid. Southern blot analyses and gene amplification experiments confirmed the integration of the transposons into the mosquito genome, although not all integrations were of the expected cut-and-paste type transposition. The increased frequency of germ-line integrations obtained with purified transposase will facilitate the generation of Mos1 transgenic mosquitoes and the application of transgenic approaches to the biology of this important vector of multiple pathogens.
Aedes↗
Insertional mutagenesis by a modified in vitro Ty1 transposition system.
Transposable elements are useful tools for insertional mutagenesis and have many potential applications in the characterization of complex genomes. Here we describe a system which facilitates the construction of large transposon insertion libraries useful for genome sequencing and functional genomic analysis. We developed two transposons, TyK and TyK'GFP+, which can be introduced into target DNAs by Ty1-mediated transposition in vitro, and several modifications which decrease the frequency of false transposition events and direct the recovery of transpositions into passenger rather than vector DNA. Insertions of TyK'GFP+ additionally may yield fusions to the Aequorea green fluorescent protein (GFP), useful in studies of gene expression and protein targeting. Transposition in vitro was obtained into target DNAs of up to 50 kb in size, restriction mapping showed insertion to be relatively random, and the sequence of 55 insertion sites showed neither strong site nor base compositional preference. Our data suggest that TyK-based artificial transposons will be suitable for a variety of genetic applications in many organisms.
DNA Transposable Elements↗
Physical mapping across the dihydrofolate reductase-thymidylate synthase chromosome of Leishmania major.
We have used a chromosome-specific approach to generate a 300 kb long 'contig' across Leishmania major 500 kb chromosome. Clones from a 13-hit genomic library served as templates to generate end-specific probes that were used in hybridization to a high density array of the library. The 'contig' generated contained 12 markers uniformly spaced. Three restriction endonucleases were mapped within the map extending its resolution. Map extension indicated a peculiar feature of sequence organization in subtelomeric regions where chromosome-specificity of mapping is lost. End-probes generated from clones mapping to the extremes of a 300 kb 'contig' rescued a high percentage of 2 types of clones from the genomic library, 1 of which showed positive hybridization to the hexameric telomere repeat. Fine mapping at these regions revealed that these 2 clones contained elements common to all chromosomes of the parasite. The physical map generated constitutes ready-to-use data for the study of many aspects of genome organization. Being cloned in a shuttle vector, the genomic sequences reordered in the map can be used to generate genetic information by transfection into the parasite.
Animals↗