RESUMEN
As part of the EULEISH international genome project, a region of 74,674 nucleotides from chromosome 21 of Leishmania major Friedlin was subcloned and sequenced; and 31 new coding sequences were predicted. Of particular interest was a unique coding strand switching region covering 1.6 kb of DNA; and this was subjected to further investigation. Bioinformatic analysis of this region revealed an unusually high AT composition, a lack of putative hairpins and a strong curvature of the DNA in agreement with the structural characteristics of similar regions of other Leishmania chromosomes. These observations and a comparison with the secondary DNA structure of four other Leishmania chromosomes and chromosomes of different organisms could suggest a functional role of this region in transcription and mitotic division.
Asunto(s)
ADN Protozoario/genética , Genes de Cambio , Leishmania major/genética , Animales , Biología Computacional , ADN Protozoario/química , Escherichia coli , Conformación de Ácido Nucleico , Análisis de Secuencia de ADN , Homología de SecuenciaRESUMEN
Lmairk, a gene encoding a member of the Aurora/Ipl1p family of protein kinases (AIRK), was cloned from the protozoan parasite Leishmania major. Aurora kinases are key enzymes involved in the regulation of normal chromosome segregation during mitosis and cytokenesis of eukaryotic cells. This single-copy gene located on L. major chromosome 28 encodes a 301 amino acid polypeptide. All 11 conserved eukaryotic protein kinase catalytic subdomains are present and the proposed AIRK signature sequence was identified in the activation loop between subdomains VII and VIII. Lmairk is expressed, as an approximately 2.4 kb message, in at least three different species of Leishmania. This report represents the first identification of an AIRK from the trypanosomatid family of early divergent eukaryotes.
Asunto(s)
Proteínas Bacterianas , Leishmania major/genética , Proteínas Quinasas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Southern Blotting , Dominio Catalítico , Mapeo Cromosómico , Clonación Molecular , ADN Protozoario , Leishmania major/enzimología , Datos de Secuencia Molecular , Proteínas Quinasas/química , Proteínas Quinasas/metabolismo , ARN Mensajero/genética , Homología de Secuencia de AminoácidoRESUMEN
The sequencing of Leishmania major Friedlin chromosome 1 (Chr1), Chr3, and Chr4 has been completed. and several other chromosomes are well underway. The complete genome sequence should be available by 2003. Over 1,000 full-length new genes have been identified, with the majority (approximately 75%) having unknown function. Many of these may be Leishmania (or kinetoplastid) specific. Most interestingly, the genes are organized into large (> 100-500 kb) polycistronic clusters of adjacent genes on the same DNA strand. Chr1 contains two such clusters organized in a "divergent" manner, i.e., the mRNAs for the two sets of genes are both transcribed towards the telomeres. Nuclear run-on analysis suggests that transcription is initiated in both directions within the "divergent" region. Chr3 and Chr4 contain two "convergent" clusters, with a single "divergent" gene at one telomere of Chr3. Sequence analysis of several genes from the LD1 region of Chr35 indicates a high degree of sequence conservation between L. major and L. donovani/L. infantum within protein-coding open reading frames (ORFs), with a lower degree of conservation within the non-coding regions. Immunization of mice with recombinant antigen from two of these genes, BTI (formerly ORFG) and ORFF, results in significant reduction in parasite burden following Leishmania challenge. Recombinant ORFF antigen shows promise as a serodiagnostic. We have also developed a tetracycline-regulated promoter system, which allows us to modulate gene expression in Leishmania.
Asunto(s)
Genoma de Protozoos , Leishmania/genética , Animales , Secuencia de Bases , Mapeo Cromosómico , Genes Protozoarios , Leishmania/clasificación , Leishmania/fisiologíaRESUMEN
The Leishmania Genome Network (LGN) was born in Rio de Janeiro, Brazil in 1994. In the short period that has elapsed since then, the LGN has focused solely on the acquisition of the resources, and hence data, that have enabled a rational approach to genomic sequencing of the reference strain, Leishmania major Friedlin. This has now been achieved. In this review, Alasdair Ivens and Jennie Blackwell, secretary and chairman of the LGN, respectively, re-examine the approaches that were adopted, comment on some of the interesting data that have been obtained and introduce some genome-wide approaches that will facilitate functional studies of the parasite.
Asunto(s)
Genoma de Protozoos , Leishmania/genética , Animales , Mapeo Cromosómico , Bases de Datos Factuales , Cooperación Internacional , Cariotipificación , Leishmania major/genética , Investigación/tendencias , Análisis de Secuencia de ADNRESUMEN
Despite the advances of modern medicine, the threat of chronic illness, disfigurement, or death that can result from parasitic infection still affects the majority of the world population, retarding economic development. For most parasitic diseases, current therapeutics often leave much to be desired in terms of administration regime, toxicity, or effectiveness and potential vaccines are a long way from market. Our best prospects for identifying new targets for drug, vaccine, and diagnostics development and for dissecting the biological basis of drug resistance, antigenic diversity, infectivity and pathology lie in parasite genome analysis, and international mapping and gene discovery initiatives are under way for a variety of protozoan and helminth parasites. These are far from ideal experimental organisms, and the influence of biological and genomic characteristics on experimental approaches is discussed, progress is reviewed and future prospects are examined.
Asunto(s)
Parásitos/genética , Animales , Bases de Datos Factuales , Eucariontes/genética , Genoma , Helmintos/genética , Humanos , Mapeo Físico de Cromosoma , Proyectos de InvestigaciónRESUMEN
The Leishmania cell surface metalloproteinase, leishmanolysin or GP63, is expressed in all stages of Leishmania major. Initial studies reported that in L. major the gp63 genes were arranged as five homologous, tandemly repeated genes (gp63 genes 1-5) and a sixth, less conserved gp63 gene located 8 kb downstream of gp63 gene 5. This study compared the sequences of L. major gp63 gene 1 and gp63 gene 6 and identified a seventh L. major gp63 gene located downstream from gp63 gene 6. The L. major gp63 genes exhibited stage-specific differences in their expression: gp63 genes 1-5 were expressed in promastigotes only, gp63 gene 6 was expressed in promastigotes and amastigotes, while gp63 gene 7 was expressed predominantly in stationary phase promastigotes and in amastigotes. Analysis of the predicted protein sequence of gp63 gene 6 (GP63-6) and gp63 gene 1 (GP63-1) showed that these two proteins were homologous in terms of overall predicted domain structure. L. major GP63-1 has been reported to contain a glycosylphosphatidylinositol (GPI) membrane anchor while sequence analysis predicted that GP63-6 contained a different hydrophobic C-terminus that may act as a transmembrane region. Transfection studies using L. major gp63 gene 1 and gp63 gene 6 expressed in L. donovani promastigotes showed that GP63-6 was expressed at the cell surface and that the distinct GP63-6 C-terminus was capable of mediating GPI anchor attachment.
Asunto(s)
Genes Protozoarios , Glicosilfosfatidilinositoles , Leishmania major/genética , Proteínas de la Membrana/genética , Metaloendopeptidasas/genética , Secuencia de Aminoácidos , Animales , Diferenciación Celular , Clonación Molecular , Expresión Génica , Leishmania major/citología , Leishmania major/enzimología , Proteínas de la Membrana/biosíntesis , Metaloendopeptidasas/biosíntesis , Datos de Secuencia Molecular , Familia de Multigenes , Procesamiento Proteico-Postraduccional , Análisis de Secuencia de ADN , Homología de Secuencia de AminoácidoRESUMEN
An extensive physical map of the Leishmania major Friedlin genome has been assembled by the combination of fingerprint analysis of a shuttle vector cosmid library and probe hybridization. The integrated data obtained for 9004 fingerprinted clones and 974 probes have placed 91.2% of the 33.58-Mb genome into contigs representing each of the 36 chromosomes. This first-generation map has already provided a suitable framework for both high-throughput DNA sequencing and functional studies of the L. major parasite.
Asunto(s)
Genoma de Protozoos , Leishmania major/genética , Mapeo Restrictivo/métodos , Animales , Dermatoglifia del ADN , Sondas de ADN , ADN Protozoario/análisis , Electroforesis en Gel de Campo Pulsado , Marcadores Genéticos , Hibridación de Ácido Nucleico , Análisis de Secuencia de ADNRESUMEN
In 1994, the World Health Organization (TDR) launched a new strategic initiative in parasite genome analysis, establishing international genome networks for filariae, Schistosoma, Leishmania, Trypanosoma brucei and T. cruzi. For Leishmania, a number of different but complementary approaches have been adopted by members of the Leishmania Genome Network. Our laboratory has been using cosmid clone fingerprinting to produce a physical map of the genome. Progress towards the completion of an integrated physical and biological map of L. major, and the preparations for genomic sequencing, are described.
Asunto(s)
Genoma de Protozoos , Leishmania major/genética , Animales , Redes de Comunicación de Computadores , Cósmidos , Dermatoglifia del ADN , Sondas de ADN , Bases de Datos Factuales , Procesamiento Automatizado de Datos , Electroforesis en Gel de Campo Pulsado , Biblioteca Genómica , Hibridación de Ácido Nucleico , Mapeo Restrictivo , Lugares Marcados de SecuenciaRESUMEN
The past few years have been significant advances in our understanding of eukaryotic genomes. In the field of parasitology, this is best exemplified by the application of genome mapping techniques to the study of genome structure and function in the protozoan parasite, Leishmania. Although much is known about the organism and the diseases it causes, molecular genetics has only recently begun to play a major part in elucidating some of the unusual characteristics of this interesting parasite. Mapping of the small (35 Mb) genome and determination of the functional role of genes by the application of in vitro homologous gene targeting techniques are revealing novel avenues for the development of prophylactic measures.
Asunto(s)
Genoma de Protozoos , Leishmania/genética , Animales , Secuencia de Bases , Mapeo Cromosómico , Cromosomas , ADN Protozoario , Humanos , Leishmania major/genética , Datos de Secuencia Molecular , Lugares Marcados de SecuenciaRESUMEN
Using a panel of patient cell lines with chromosomal breakpoints, we constructed a physical map for the short arm of human chromosome 11. We focused on 11p15, a chromosome band harboring at least 25 known genes and associated with the Beckwith-Wiedemann syndrome, several childhood tumors, and genomic imprinting. This underlines the need for a physical map for this region. We divided the short arm of chromosome 11 into 18 breakpoint regions, and a large series of new and previously described genes and markers was mapped within these intervals using fluorescence in situ hybridization. Cosmid fingerprint analysis showed that 19 of these markers were included in cosmid contigs. A detailed 10-Mb pulsed-field physical map of the region 11p15.3-pter was constructed. These three different approaches enabled the high-resolution mapping of 210 markers, including 22 known genes.
Asunto(s)
Aberraciones Cromosómicas , Trastornos de los Cromosomas , Cromosomas Humanos Par 11 , Síndrome de Beckwith-Wiedemann/genética , Southern Blotting , Línea Celular , Niño , Mapeo Cromosómico , Cósmidos , Marcadores Genéticos , Impresión Genómica , Humanos , Hibridación Fluorescente in Situ , Neoplasias/genética , Mapeo RestrictivoRESUMEN
We describe progress in a continuing project aimed at the generation of an overlapping cosmid DNA clone map of the short arm of human chromosome 11. The automated procedures used to prepare DNA samples and the computerized data collection and recording systems are described. We also demonstrate the use of the clones as reagents for the rapid isolation of genomic DNAs containing smaller probed regions. We have isolated approximately 4700 human cosmid DNA clones from mouse/human hybrid cell lines that contain predominantly human chromosomal region 11p. Of the DNA in the cell lines, 60% is derived from this chromosomal region, and the remaining 40% is derived from regions of chromosomes 3, 19, and 20. A total of 4159 clones have been fingerprinted to identify potential overlaps, and we have developed 535 sets ("contigs"). Using random modeling, it is estimated that 65% of 11p must be contained in the analyzed cosmids. The database of clones has been used to identify single or overlapping clones from noncosmid DNA probes. Examples are presented. It is proposed that cosmid reference filters be distributed to requesting laboratories.