RESUMO
The precise regulation of growth factor signalling is crucial to the molecular control of development in Drosophila. Post-translational modification of signalling molecules is one of the mechanisms that modulate developmental signalling specificity. We describe a new gene, fringe connection (frc), that encodes a nucleotide-sugar transporter that transfers UDP-glucuronic acid, UDP-N-acetylglucosamine and possibly UDP-xylose from the cytoplasm into the lumen of the endoplasmic reticulum/Golgi. Embryos with the frc mutation display defects in Wingless, Hedgehog and fibroblast growth factor signalling. Clonal analysis shows that fringe-dependent Notch signalling is disrupted in frc mutant tissue.
Assuntos
Drosophila melanogaster/genética , Glicosiltransferases/metabolismo , Heparitina Sulfato/metabolismo , N-Acetilglucosaminiltransferases , Transdução de Sinais/fisiologia , Sequência de Aminoácidos , Animais , Citoplasma/metabolismo , Proteínas de Drosophila , Drosophila melanogaster/embriologia , Drosophila melanogaster/crescimento & desenvolvimento , Retículo Endoplasmático/metabolismo , Glicosiltransferases/genética , Complexo de Golgi/metabolismo , Humanos , Dados de Sequência Molecular , Morfogênese , Fenótipo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Uridina Difosfato Ácido Glucurônico/metabolismo , Uridina Difosfato N-Acetilglicosamina/metabolismo , Uridina Difosfato Xilose/metabolismo , Asas de Animais/embriologia , Asas de Animais/crescimento & desenvolvimentoRESUMO
Transposable elements of the mariner/Tc1 family are postulated to have spread by horizontal transfer and be relatively independent of host-specific factors. This was tested by introducing the Drosophila mauritiana element mariner into the human parasite Leishmania major, a trypanosomatid protozoan belonging to one of the most ancient eukaryotic lineages. Transposition in Leishmania was efficient, occurring in more than 20 percent of random transfectants, and proceeded by the same mechanism as in Drosophila. Insertional inactivation of a specific gene was obtained, and a modified mariner element was used to select for gene fusions, establishing mariner as a powerful genetic tool for Leishmania and other organisms. These experiments demonstrate the evolutionary range of mariner transposition in vivo and underscore the ability of this ubiquitous DNA to parasitize the eukaryotic genome.
Assuntos
Cinamatos , DNA Nucleotidiltransferases/genética , Elementos de DNA Transponíveis , Drosophila/genética , Leishmania major/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Evolução Biológica , DNA Nucleotidiltransferases/química , Resistência a Medicamentos , Genes de Protozoários , Genoma de Protozoário , Higromicina B/análogos & derivados , Higromicina B/farmacologia , Leishmania major/efeitos dos fármacos , Mutagênese Insercional , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Protozoário/genética , RNA de Protozoário/metabolismo , Especificidade da Espécie , Transfecção , TransposasesRESUMO
For virulence and transmission, the protozoan parasite Leishmania must assemble a complex glycolipid on the cell surface, the lipophosphoglycan (LPG). Functional complementation identified the gene LPG2, which encodes an integral Golgi membrane protein implicated in intracellular compartmentalization of LPG biosynthesis. Ipg2- mutants lack only characteristic disaccharide-phosphate repeats, normally present on both LPG and other surface or secreted molecules considered critical for infectivity. In contrast, a related yeast gene, VAN2/VRG4, is essential and required for general Golgi function. These results suggest that LPG2 participates in a specialized virulence pathway, which may offer an attractive target for chemotherapy.
Assuntos
Genes de Protozoários , Glicoesfingolipídeos/biossíntese , Glicoesfingolipídeos/fisiologia , Complexo de Golgi/metabolismo , Leishmania donovani/patogenicidade , Proteínas de Membrana/fisiologia , Proteínas de Protozoários/fisiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Sequência de Carboidratos , Compartimento Celular , Genes Fúngicos , Teste de Complementação Genética , Glicoesfingolipídeos/química , Glicoesfingolipídeos/genética , Glicosilação , Leishmania donovani/genética , Leishmania donovani/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/genética , Dados de Sequência Molecular , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Virulência/genética , Leveduras/genéticaRESUMO
A critical step in the infectious cycle of Leishmania is the differentiation of parasites within the sand fly vector to the highly infective metacyclic promastigote stage. Here, we establish tetrahydrobiopterin (H4B) levels as an important factor controlling the extent of metacyclogenesis. H4B levels decline substantially during normal development, and genetic or nutritional manipulations showed that low H4B caused elevated metacyclogenesis. Mutants lacking pteridine reductase 1 (PTR1) had low levels of H4B, remained infectious to mice, and induced larger cutaneous lesions (hypervirulence). Thus, the control of pteridine metabolism has relevance to the mechanism of Leishmania differentiation and the limitation of virulence during evolution.
Assuntos
Biopterinas/análogos & derivados , Biopterinas/metabolismo , Leishmania major/crescimento & desenvolvimento , Leishmania major/metabolismo , Leishmaniose Cutânea/parasitologia , Proteínas de Membrana Transportadoras , Proteínas de Protozoários , Animais , Biopterinas/farmacologia , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Cromatografia Líquida de Alta Pressão , Ácido Fólico/metabolismo , Genes de Protozoários , Glicoesfingolipídeos/análise , Leishmania major/genética , Leishmania major/patogenicidade , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Mutação , Oxirredutases/genética , Oxirredutases/metabolismo , Transdução de Sinais , VirulênciaRESUMO
We have examined the transcriptional organization of the R region of the protozoan parasite Leishmania major. This region encodes the bifunctional enzyme dihydrofolate reductase-thymidylate synthase (DHFR-TS) and is frequently amplified as a 30-kilobase (kb) extrachromosomal circular DNA in methotrexate-resistant lines. Northern (RNA) blot analysis shows that the R region encodes at least 10 stable cytoplasmic polysomal poly(A)+ RNAs, ranging in size from 1.7 to 13 kb and including the 3.2-kb DHFR-TS mRNA. Transcriptional mapping reveals that these RNAs are closely spaced and collectively cover more than 95% of the 30-kb amplified R region. The organization is complex, including several overlapping RNAs 3' of DHFR-TS and two examples of antisense RNAs 5' of DHFR-TS. The R region RNAs can be grouped into two empirical domains, with eight contiguous RNAs transcribed in the same direction as that of DHFR-TS and two contiguous RNAs transcribed in the orientation opposite to that of DHFR-TS. The two 5'-most RNAs of the DHFR-TS-containing domain overlap the RNAs transcribed from the opposite strand. These data are relevant to models of transcription, including recent studies suggesting polycistronic transcription in trypanosomatids. The abundance of R region RNAs increases uniformly 10- to 15-fold in the amplified R1000-3 line relative to the wild type, and no new RNAs were observed. This suggests that all elements required in cis for DHFR-TS expression are contained within the 30-kb circular DNA. Quantitative analysis reveals that the steady-state DHFR-TS mRNA and protein levels are not growth phase regulated, unlike the monofunctional mouse DHFR. DHFR-TS is developmentally regulated, however, declining about fivefold in lesion amastigotes relative to promastigotes.
Assuntos
Leishmania tropica/genética , Tetra-Hidrofolato Desidrogenase/genética , Timidilato Sintase/genética , Animais , Mapeamento Cromossômico , Amplificação de Genes , Leishmania tropica/crescimento & desenvolvimento , Leishmania tropica/metabolismo , Polirribossomos/metabolismo , RNA/genética , RNA/metabolismo , RNA Antissenso , RNA Mensageiro/genética , Homologia de Sequência do Ácido Nucleico , Transcrição GênicaRESUMO
We describe the structure of amplified DNA that was discovered in two laboratory stocks of the protozoan parasite Leishmania tarentolae. Restriction mapping and molecular cloning revealed that a region of 42 kilobases was amplified 8- to 30-fold in these lines. Southern blot analyses of digested DNAs or chromosomes separated by pulsed-field electrophoresis showed that the amplified DNA corresponded to the H region, a locus defined originally by its amplification in methotrexate-resistant Leishmania major (S. M. Beverley, J. A. Coderre, D. V. Santi, and R. T. Schimke, Cell 38:431-439, 1984). Similarities between the amplified DNA of the two species included (i) extensive cross-hybridization; (ii) approximate conservation of sequence order; (iii) extrachromosomal localization; (iv) an overall inverted, head-to-head configuration as a circular 140-kilobase tetrameric molecule; (v) two regions of DNA sequence rearrangement, each of which was closely associated with the two centers of the inverted repeats; (vi) association with methotrexate resistance; and (vii) phenotypically conservative amplification, in which the wild-type chromosomal arrangement was retained without apparent modification. Our data showed that amplified DNA mediating drug resistance arose in unselected L. tarentolae, although the pressures leading to apparently spontaneous amplification and maintenance of the H region are not known. The simple structure and limited extent of DNA amplified in these and other Leishmania lines suggests that the study of gene amplification in Leishmania spp. offers an attractive model system for the study of amplification in cultured mammalian cells and tumors. We also introduced a method for measuring the size of large circular DNAs, using gamma-irradiation to introduce limited double-strand breaks followed by sizing of the linear DNAs by pulsed-field electrophoresis.
Assuntos
DNA Circular/genética , Amplificação de Genes , Leishmania tropica/genética , Leishmania/genética , Animais , Southern Blotting , DNA Circular/efeitos da radiação , Resistência a Medicamentos/genética , Leishmania tropica/efeitos dos fármacos , Metotrexato/farmacologia , Mapeamento por RestriçãoRESUMO
The genome of the trypanosomatid protozoan genus Leishmania has been shown to undergo a number of changes relevant to drug resistance and virulence, such as gene amplification, chromosomal rearrangement, and variation in ploidy. Experimental approaches to the study of genomic changes have in some cases been limited by the fact that Leishmania cells are asexual diploids, as are some other trypanosomatids, pathogenic fungi, and cultured mammalian cells. Here we report upon a system which permits the measurement of several types of genomic change occurring at the dihydrofolate reductase-thymidylate synthase (DHFR-TS) locus. First, we show that DHFR-TS can function as a positive/negative marker. We used selection against DHFR-TS on a heterozygous line (+/HYG) to generate colonies exhibiting both loss of heterozygosity and structural mutations in DHFR-TS, permitting the first measurement of mutation frequencies in this parasite. Loss of heterozygosity occurred at a frequency ranging from 10(-4) to 10(-6) and was elevated 24-fold by treatment with gamma-irradiation, while the frequency of other events was less than 10(-6) and was increased more than 1,000-fold by nitrosoguanidine treatment. The frequency of loss of heterozygosity relative to other processes such as mutation and gene replacement has important implications for genetic variability in natural Leishmania populations and the generation of both targeted and random mutations. We also developed a protocol for null targeting of diploid cells, in which transfection of a DHFR-TS deletion construct into Leishmania cells followed by negative selection yielded parasites lacking DHFR-TS or foreign sequences. The null-targeting method can be applied to any diploid cell, at any locus for which a negative selection exists. Such marker-free auxotrophic Leishmania cells show potential as an attenuated vaccine, and the methods developed here provide a new approach for manipulating and characterizing the plasticity of the Leishmania genome.
Assuntos
Deleção Cromossômica , Genes de Protozoários , Leishmania major/enzimologia , Leishmania major/genética , Tetra-Hidrofolato Desidrogenase/genética , Timidilato Sintase/genética , Animais , Mapeamento Cromossômico , DNA de Protozoário/química , DNA de Protozoário/metabolismo , Resistência a Medicamentos/genética , Marcadores Genéticos , Leishmania major/patogenicidade , Metotrexato , Metilnitronitrosoguanidina , Mutagênese , Ploidias , Reação em Cadeia da Polimerase , Seleção Genética , VirulênciaRESUMO
Murine 3T6 selected in increasing concentrations of methotrexate were unstable with respect to dihydrofolate reductase overproduction and methotrexate resistance when they are cultured in the absence of methotrexate. An analysis of the karyotypes of these resistant cells revealed the presence of numerous double minute chromosomes. We observed essentially identical kinetics of loss of dihydrofolate reductase gene sequences in total deoxyribonucleic acid and in deoxyribonucleic acid from fractions enriched in double minute chromosomes and in the numbers of double minute chromosomes per cell during reversion to methotrexate sensitivity, and this suggested that unstably amplified gene sequences were localized on double minute chromosomes. This conclusion ws also supported by an analysis of cell populations sorted according to dihydrofolate reductase enzyme contents, in which relative gene amplification and double minute chromosome content were related proportionally.
Assuntos
Aberrações Cromossômicas , Amplificação de Genes , Tetra-Hidrofolato Desidrogenase/genética , Animais , Linhagem Celular , Enzimas de Restrição do DNA/farmacologia , Desoxirribonuclease EcoRI , Resistência a Medicamentos , Herança Extracromossômica/efeitos dos fármacos , Fibroblastos , Citometria de Fluxo , Cariotipagem , Metotrexato/farmacologia , Camundongos , Camundongos EndogâmicosRESUMO
To delineate segments of the genome of the human protozoan parasite Leishmania major necessary for replication and expression, we developed a vector (pR-NEO) which can be reproducibly introduced into L. major. This DNA was derived from a 30-kilobase extrachromosomal amplified DNA bearing the dihydrofolate reductase-thymidylate synthase gene, with the coding region for neomycin phosphotransferase substituted for that of dihydrofolate reductase-thymidylate synthase and a bacterial origin of replication and selectable marker added. G418-resistant lines were obtained at high efficiency by electroporation of pR-NEO (approaching 10(-4) per cell), while constructs bearing an inverted neo gene or lacking Leishmania sequences did not confer resistance. pR-NEO replicated in L. major and gave rise to correctly processed transcripts bearing the trans-spliced miniexon. Molecular karyotype analysis showed that in some lines pR-NEO DNA exists exclusively as an extrachromosomal circle, a finding supported by the rescue of intact pR-NEO after transformation of Escherichia coli. These data genetically localize all elements required in cis for DNA replication, transcription, and trans splicing to the Leishmania DNA contained within pR-NEO DNA and signal the advent of stable transfection methodology for addressing molecular phenomena in trypanosomatid parasites.
Assuntos
Vetores Genéticos , Leishmania tropica/genética , Animais , Sequência de Bases , Northern Blotting , Replicação do DNA , DNA Recombinante , Herança Extracromossômica , Dados de Sequência Molecular , Transcrição Gênica , TransfecçãoRESUMO
We describe the first example of unstable gene amplification consisting of linear extrachromosomal DNAs in drug-resistant eukaryotic cells. alpha-Difluoromethylornithine (DFMO)-resistant Leishmania donovani with an amplified ornithine decarboxylase (ODC) gene copy number contained two new extrachromosomal DNAs, both present in 10 to 20 copies. One of these was a 140-kb linear DNA (ODC140-L) on which all of the amplified copies of the odc gene were located. The second was a 70-kb circular DNA (ODC70-C) containing an inverted repeat but lacking the odc gene. Both ODC140-L and ODC70-C were derived from a preexisting wild-type chromosome, probably by a conservative amplification mechanism. Both elements were unstable in the absence of DFMO, and their disappearance coincided with a decrease in ODC activity and an increase in DFMO growth sensitivity. These results suggest the possibility that ODC70-C may play a role in DFMO resistance. These data expand the diversity of known amplification mechanisms in eukaryotes to include the simultaneous unstable amplification of both linear and circular DNAs. Further characterization of these molecules will provide insights into the molecular mechanisms underlying gene amplification, including the ability of linear amplified DNAs to acquire telomeres and the determinants of chromosomal stability.
Assuntos
Eflornitina/farmacologia , Amplificação de Genes , Leishmania donovani/genética , Ornitina Descarboxilase/genética , Animais , Southern Blotting , Sondas de DNA , Resistência a Medicamentos/genética , Eletroforese em Gel de Campo Pulsado , Exodesoxirribonucleases/metabolismo , Herança Extracromossômica , Raios gama , Genes de Protozoários , Concentração de Íons de Hidrogênio , Leishmania donovani/efeitos dos fármacos , Leishmania donovani/enzimologia , Ornitina Descarboxilase/metabolismo , Ornitina Descarboxilase/efeitos da radiação , Homologia de Sequência , Telômero , Proteínas ViraisRESUMO
Glycosomes are membrane-bounded microbody organelles that compartmentalize glycolysis as well as other important metabolic processes in trypanosomatids. The compartmentalization of these enzymatic reactions is hypothesized to play a crucial role in parasite physiology. Although the metabolic role of glycosomes differs substantially from that of the peroxisomes that are found in other eukaryotes, similarities in signals targeting proteins to these organelles suggest that glycosomes and peroxisomes may have evolved from a common ancestor. To examine this hypothesis, as well as gain insights into the function of the glycosome, we used a positive genetic selection procedure to isolate the first Leishmania mutant (gim1-1 [glycosome import] mutant) with a defect in the import of glycosomal proteins. The mutant retains glycosomes but mislocalizes a subset glycosomal proteins to the cytoplasm. Unexpectedly, the gim1-1 mutant lacks lipid bodies, suggesting a heretofore unknown role of the glycosome. We used genetic approaches to identify a gene, GIM1, that is able to restore import and lipid bodies. A nonsense mutation was found in one allele of this gene in the mutant line. The predicted Gim1 protein is related the peroxin 2 family of integral membrane proteins, which are required for peroxisome biogenesis. The similarities in sequence and function provide strong support for the common origin model of glycosomes and peroxisomes. The novel phenotype of gim1-1 and distinctive role of Leishmania glycosomes suggest that future studies of this system will provide a new perspective on microbody biogenesis and function.
Assuntos
Genes de Protozoários/fisiologia , Leishmania donovani/genética , Proteínas de Membrana/genética , Microcorpos , Proteínas de Protozoários/genética , Sequência de Aminoácidos , Animais , Dosagem de Genes , Genes de Protozoários/genética , Leishmania donovani/ultraestrutura , Microcorpos/metabolismo , Microcorpos/ultraestrutura , Dados de Sequência Molecular , Fator 2 da Biogênese de Peroxissomos , Filogenia , Proteínas de Protozoários/metabolismo , RNA Mensageiro/análise , RNA de Protozoário/análise , Mapeamento por Restrição , Análise de Sequência de DNA , Homologia de Sequência de AminoácidosRESUMO
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.
Assuntos
Elementos de DNA Transponíveis/genética , Evolução Molecular , Genoma , Sequências Reguladoras de Ácido Nucleico/genética , Transativadores/fisiologia , Transposases/metabolismo , Animais , Elementos de DNA Transponíveis/fisiologia , DNA de Protozoário/química , DNA de Protozoário/genética , DNA de Protozoário/metabolismo , DNA Super-Helicoidal/química , DNA Super-Helicoidal/genética , DNA Super-Helicoidal/metabolismo , Proteínas de Ligação a DNA/genética , Drosophila/enzimologia , Drosophila/genética , Magnésio/metabolismo , Manganês/metabolismo , Mutagênese Insercional/métodos , Plasmídeos/química , Plasmídeos/genética , Plasmídeos/metabolismo , Dobramento de Proteína , Renaturação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Recombinação Genética/genética , Deleção de Sequência/genética , Especificidade por Substrato , Sequências Repetidas Terminais/genética , Transposases/química , Transposases/genética , Transposases/isolamento & purificaçãoRESUMO
Leishmania exploits several strategies to survive within the phagolysosome of vertebrate macrophages and be transmitted by sand fly vectors. Recent advances in functional genetic analysis provide a new avenue for identifying genes implicated in the infectious cycle of the parasite, such as those necessary for the synthesis and expression of the key surface glycoconjugate, lipophosphoglycan (LPG).
Assuntos
Glicoesfingolipídeos/genética , Leishmania/genética , Leishmania/patogenicidade , Virulência/genética , Animais , Sequência de Carboidratos , Genes de Protozoários , Glicoesfingolipídeos/análise , Leishmania/química , Leishmania/crescimento & desenvolvimento , Dados de Sequência Molecular , Saccharomyces cerevisiae/genética , Relação Estrutura-AtividadeRESUMO
Lipophosphoglycan is a prominent member of the phosphoglycan-containing surface glycoconjugates of Leishmania. Genetic tests enable confirmation of its role in parasite virulence and permit discrimination between the roles of lipophosphoglycan and related glycoconjugates. When two different lipophosphoglycan biosynthetic genes from Leishmania major were knocked out, there was a clear loss of virulence in several steps of the infectious cycle but, with Leishmania mexicana, no effect on virulence was found. This points to an unexpected diversity in the reliance of Leishmania species on virulence factors, a finding underscored by recent studies showing great diversity in the host response to Leishmania species.
Assuntos
Glicoesfingolipídeos/fisiologia , Leishmania/patogenicidade , Animais , Glicoesfingolipídeos/biossíntese , Glicoesfingolipídeos/genética , Humanos , Insetos Vetores/parasitologia , Leishmania/genética , Leishmania major/genética , Leishmania major/patogenicidade , Leishmania mexicana/genética , Leishmania mexicana/patogenicidade , Leishmaniose/parasitologia , Leishmaniose/fisiopatologia , Macrófagos/parasitologia , Camundongos , Psychodidae/parasitologia , VirulênciaRESUMO
We have developed two shuttle cosmid vectors for the trypanosomatid protozoan parasite Leishmania. Cosmids cLHYG and cLNEO contain hyg and neo markers, conferring resistance to hygromycin B and G418, respectively, replicate extrachromosomally after transfection into promastigotes, and bear a unique BamHI cloning site. To ensure the representation of telomeric sequences, which represent about 5% of the Leishmania genome, random insert DNAs were prepared by shearing followed by blunt-end ligation with BamHI adapters. Representative genomic libraries from Leishmania species representing the four major pathogenic complexes were prepared using cosmid cLHYG. The cosmid libraries were efficiently transfected into Leishmania, and individual cosmids were readily recovered by transformation back into Escherichia coli. The relatively small size of the Leishmania genome (50 Mb) combined with the capacity and transfection efficiency of these cosmid libraries (> 1000 Leishmania transfectants/plate) suggests the feasibility of functional genetic complementation in this parasite.
Assuntos
Cosmídeos , Genes de Protozoários , Biblioteca Genômica , Leishmania/genética , Transfecção/métodos , Animais , Sequência de Bases , Clonagem Molecular , DNA de Protozoário/genética , DNA Recombinante , Desoxirribonuclease BamHI , Resistência a Medicamentos/genética , Escherichia coli , Teste de Complementação Genética , Marcadores Genéticos , Gentamicinas/farmacologia , Higromicina B/farmacologia , Dados de Sequência Molecular , Mutagênese Insercional , Especificidade da Espécie , TelômeroRESUMO
We describe a transient transfection protocol for cultured Leishmania major promastigotes, utilizing Escherichia coli genes encoding beta-galactosidase and beta-glucuronidase inserted into an expression vector derived from the dihydrofolate reductase-thymidylate synthase locus. Less than 0.1 pg of either reporter enzyme can be detected with a simple fluorimetric assay, and transfection of 10 micrograms of either reporter construct yields activities at least 100-fold over background. Simultaneous introduction of both constructs showed that the activity of each reporter gene was unaffected by the presence of the other, allowing one reporter construct to serve as a control for experimental variability in test gene constructs containing the second reporter gene. These results show that it is feasible to apply transient expression assays to the identification of cis-acting elements of genes encoding nonabundant mRNAs in the genus Leishmania.
Assuntos
Expressão Gênica/fisiologia , Leishmania tropica/genética , Transfecção , Animais , Escherichia coli/genética , Vetores Genéticos/genética , Glucuronidase/genética , Cinética , Leishmania tropica/fisiologia , Plasmídeos/genética , Tetra-Hidrofolato Desidrogenase/genética , beta-Galactosidase/genéticaRESUMO
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.
Assuntos
Elementos de DNA Transponíveis/genética , Mutagênese Insercional/genética , Animais , Sequência de Bases , Replicação do DNA/genética , Drosophila/genética , Resistência Microbiana a Medicamentos/genética , Escherichia coli/genética , Marcadores Genéticos , Proteínas de Fluorescência Verde , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Dados de Sequência Molecular , Neomicina/farmacologia , Plasmídeos/genética , Biossíntese de Proteínas , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
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.
Assuntos
Elementos de DNA Transponíveis , Vetores Genéticos , Mutagênese Insercional , Saccharomyces cerevisiae/genética , DNA Fúngico/genética , Escherichia coli/genética , Transformação GenéticaRESUMO
We have utilized pulsed field electrophoresis to characterize several karyotypic alterations in Leishmania major. Promastigotes of the LT252 line contain three small chromosomes, of 300, 350 and 385 kb. Quantitative densitometry of ethidium bromide-stained gels suggest that these chromosomes are present in equal levels (2:2:2). Two derivatives of this line, one appearing spontaneously (LT252 delta) and one obtained following selection with methotrexate (11-MTXR20), exhibit altered levels of these chromosomes, in the ratio of 2:1:3, respectively. The variant pattern in both lines is due to an increase in size of chromosome 2, yielding a new chromosome similar in size to chromosome 3. The enlarged chromosome 2 of the LT252 delta line is a result of amplification of the mini-exon gene array normally located on this chromosome, which increases from about 93 to 150 copies of the 0.44-kb mini-exon tandem repeat, as shown by quantitative hybridization and sizing of the mini-exon array. In contrast, the increased size of chromosome 2 within the methotrexate-resistant mutant 11-MTXR20 is not due to mini-exon amplification. In both variant lines, there are equal levels of the wild-type and enlarged chromosome 2, and the wild-type chromosome 2 is now present at 50% of the level of chromosome 1. These and other data suggest that Leishmania is diploid for chromosomes bearing housekeeping genes such as the mini-exon locus.
Assuntos
Cromossomos/ultraestrutura , Diploide , Amplificação de Genes , Variação Genética , Leishmania tropica/genética , Animais , Southern Blotting , Resistência a Medicamentos , Eletroforese/métodos , Éxons , Leishmania tropica/efeitos dos fármacos , Metotrexato/farmacologiaRESUMO
Genetic transformation of Leishmania has relied upon two exogenous selectable markers, neo and hyg, encoding resistance to G418 and hygromycin B respectively. There is a need for multiple independent selectable markers, since Leishmania is diploid and experimental sexual crosses are not currently feasible. Here we report on the development of two additional markers: pac, conferring resistance to the glycopeptide antibiotic puromycin, and phleo, conferring resistance to the DNA-binding drug phleomycin. We constructed a set of four analogous shuttle vectors with these four markers, using DNA segments flanking the Leishmania major H region hmtxr gene to provide information required for expression. These constructs (pHM-NEO, pHM-HYG, pHM-PAC and pHM-PHLEO) were successfully transfected into L. major, mostly with efficiencies comparable to those observed with previous DHFR-TS-based neo and hyg-containing constructs. The exception was pHM-PHLEO, which transfected 30-fold less efficiently; this may be related to the nonenzymatic mechanism of resistance encoded by phleo. All four constructs were shown to replicate extra-chromosomally. Stable transfectants bearing all paired combinations of pHM constructs were obtained by a second round of transfection. These data show that the four markers are functionally independent and in conjunction with the Leishmania N-acetylglucosaminyl transferase gene, brings the number of selectable markers available in Leishmania to five.