RESUMO
We hereby present the complete sequence and annotation of pRG930cm, a spectinomycin/streptomycin/chloramphenicol-resistant cosmid vector. pRG930cm (17,256 bp; GenBank Accession No.: FM174471) has a broad host range, and is stably maintained by a number of Gram-negative bacteria including Pseudomonas spp, Escherichia coli, Agrobacterium tumefaciens and Azorhizobium caulinodans ORS571. pRG930cm is already widely used and its sequence will aid efficient construction and analysis of cosmid libraries.
Assuntos
Azorhizobium caulinodans/genética , Cosmídeos , Escherichia coli/genética , Pseudomonas/genética , Agrobacterium tumefaciens/genética , Resistência ao Cloranfenicol , Engenharia Genética , Espectinomicina , EstreptomicinaRESUMO
We report the cloning of a Trypanosoma cruzi gene encoding a solanesyl-diphosphate synthase, TcSPPS. The amino acid sequence (molecular mass approximately 39 kDa) is homologous to polyprenyl-diphosphate synthases from different organisms, showing the seven conserved motifs and the typical hydrophobic profile. TcSPPS preferred geranylgeranyl diphosphate as the allylic substrate. The final product, as determined by TLC, had nine isoprene units. This suggests that the parasite synthesizes mainly ubiquinone-9 (UQ-9), as described for Trypanosoma brucei and Leishmania major. In fact, that was the length of the ubiquinone extracted from epimastigotes, as determined by high-performance liquid chromatography. Expression of TcSPPS was able to complement an Escherichia coli ispB mutant. A punctuated pattern in the cytoplasm of the parasite was detected by immunofluorescence analysis with a specific polyclonal antibody against TcSPPS. An overlapping fluorescence pattern was observed using an antibody directed against the glycosomal marker pyruvate phosphate dikinase, suggesting that this step of the isoprenoid biosynthetic pathway is located in the glycosomes. Co-localization in glycosomes was confirmed by immunogold electron microscopy and subcellular fractionation. Because UQ has a central role in energy production and in reoxidation of reduction equivalents, TcSPPS is promising as a new chemotherapeutic target.
Assuntos
Alquil e Aril Transferases/biossíntese , Microcorpos/metabolismo , Trypanosoma cruzi/metabolismo , Alquil e Aril Transferases/química , Sequência de Aminoácidos , Animais , Cromatografia em Camada Fina , Clonagem Molecular , Cosmídeos , Escherichia coli/metabolismo , Teste de Complementação Genética , Mitocôndrias/metabolismo , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Ubiquinona/química , Ubiquinona/isolamento & purificaçãoRESUMO
A two-year-old boy presenting with bilateral aniridia and psychomotor retardation had a de novo (2;3;11) highly complex rearrangement which was characterized as far as possible by means of G-banding and FISH assays with multiple probes including cosmids for the Wilms, Aniridia, Genital anomalies and Retardation (WAGR) region, alphoid repeats for chromosomes 2, 3 and 11, subtelomere probes for 2p/2q, 3p/3q and 11q and BACs for 2q32 and 3q13. We identified approximately 15 breakpoints with at least three interchromosomal and three intrachromosome anomalies involving chromosome 11. Both parents had normal karyotypes and no cryptic 11p rearrangements revealed by the chromosome 11 cosmid panel. The lack of a deletion of PAX6 pointed to the direct insertion of an approximately 300-kb segment involving the cosmids FO2121 and AO4160, and more specifically the insertion's proximal breakpoint in the approximately 150-kb segment between FO2121 and FAT5 (PAX6), as the responsible factor for the patient's aniridia via a position effect resulting in functional haploinsufficiency of the PAX6 gene. This case illustrates the importance of recognizing that de novo complex chromosomal rearrangements found in patients with diverse clinical features may contribute to the phenotype, but that multiple mechanisms and higher levels of complexity may be unmasked by high resolution molecular cytogenetic studies.
Assuntos
Aniridia/genética , Cromossomos Humanos Par 11 , Cromossomos Humanos Par 2 , Cromossomos Humanos Par 3 , Pré-Escolar , Bandeamento Cromossômico , Mapeamento Cromossômico , Cosmídeos , Elementos de DNA Transponíveis , Lateralidade Funcional , Rearranjo Gênico , Humanos , Hibridização in Situ Fluorescente , Cariotipagem , Masculino , Fator de Transcrição PAX5/genética , ProibitinasRESUMO
The human immunoglobulin lambda locus ( IGL) is mapped at Chromosome 22q11.2, spanning about 1 Mb of DNA, and directs the synthesis of lambda-type immunoglobulin light chains. The positions of about 73-74 germline V-lambda genes, depending on the haplotypes, are known, with 29-33 of them being functional IGLV genes. These genes were divided into 11 subgroups ( IGLV1 to IGLV11) distributed into three gene clusters ( VA, VB, and VC). We constructed a high-resolution restriction map of a 37-kb cosmid clone (cosmid 8.3) harboring genes of the IGLV1, IGLV7, and IGLV5 families and the non-coding sequences IGLV(I)-42 and IGLV(VII)-41-1, located at cluster VB of the IGL locus. These IGLV genes were associated with unique EcoRI fragments detectable in Southern blots of genomic DNA. Population RFLP has revealed new IGLV alleles and haplotypes. We used the restriction map of cosmid 8.3 and the IMGT database as a reference for RFLP studies. EcoRI Southern blot hybridizations with subgroup-specific probes of the functional and open reading frame sequences present in cosmid 8.3 revealed different frequencies of IGLV gene fragments, as well as deletions of IGLV1-50 and IGLV5-39 genes and RFLP involving IGLV5-45 and IGLV5-48 genes. All members of the IGLV7 subgroup were monomorphic. Sequencing of the genes present in cosmid 8.3 revealed a new allelic variant of the IGLV5 subgroup. These data contribute to a better understanding of the contribution of the germline IGLV genes to the human genetic background and polymorphism.
Assuntos
Cromossomos Humanos Par 22/genética , Cosmídeos , Desoxirribonuclease EcoRI/genética , Região Variável de Imunoglobulina/genética , Cadeias lambda de Imunoglobulina/genética , Polimorfismo Genético , Alelos , Sequência de Aminoácidos , Sequência de Bases , Biomarcadores , Southern Blotting , Elementos de DNA Transponíveis , Deleção de Genes , Frequência do Gene , Genômica , Humanos , Dados de Sequência Molecular , Polimorfismo de Fragmento de RestriçãoRESUMO
The feasibility of genetic manipulation in trypanosomatids has allowed the introduction of molecular approaches for the investigation of gene function. The development of cosmids that carry approximately 40 kb of insert and can be easily introduced in trypanosomatids greatly increased the possibility of gene rescue by functional complementation in these parasites. Although functional complementation is widely used, some of its aspects, such as differential levels of expression along the insert clone, are not clear. We have used the DHFRTS gene as a tool to better understand the mechanisms of transcription of genes present in episomes and the results obtained via functional complementation in Leishmania. This gene was chosen not only because its inactivation in the parasite generates an easily recoverable phenotype, auxotrophy for thymidine (TdR), but also because null mutants are already available. The null mutant available contained two resistance markers (neomycin phosphotransferase-NEO and hygromycin phosphotransferase-HYG), and the loss of heterozygosity (LOH) was induced to recover clones sensitive to hygromycin B, which were necessary for the rescue of transfectants. Analyses of the Leishmania clones confirmed the loss of the HYG gene associated with unanticipated genomic rearrangements. A LOH clone was transfected with cosmids containing the DHFRTS gene in several distinct contexts in order to evaluate the levels of expression of the complementing gene. Results presented here show that the lost phenotype is rescued, irrespective to the DHFRTS location or direction of transcription indicating that functional complementation can be achieved without concern for the position of the complementing gene in a cosmid.
Assuntos
Leishmania/genética , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , Plasmídeos/genética , Mapeamento por Restrição , Tetra-Hidrofolato Desidrogenase/genética , Tetra-Hidrofolato Desidrogenase/metabolismo , Timidilato Sintase/genética , Timidilato Sintase/metabolismo , Animais , Cosmídeos , Teste de Complementação Genética , Leishmania/crescimento & desenvolvimento , Leishmania/metabolismo , Perda de Heterozigosidade , Mutação , Fenótipo , Recombinação Genética , Transcrição Gênica , TransfecçãoRESUMO
L'analyse génomique de Mycobacterium leprae, l'agent pathogène de la lèpre, a été réalisée grâce au séquençage à haut débit des cosmides et plasmides préparés à partir de l'ADN d´une souche isolée d'un patient. Elle a montré que ce bacille possède un seul chromosome circulaire, dont la taille (3,2Mb) et la composition en G+C (57,8%) sont réduits par rapport aux austres chromosomes mycobactériens connus. L'analyse informatique de la séquence a mis en évidence que seule la moitié de la séquences est codante. L'autre moitié contient des pseudogènes et des séquences non codantes. Le génome de M. leprae a donc subi une évolution réductive très importante qui ne lui a laissé qu'un set minimun de gènes fonctionnels pour vivre. Les informations déduites de la partie codante de la séquence permettent d'appréhender les propriétés biologiques particulières de cet agent infectieux à développement intra-cellulaire obligatoire. De plus, la disparition de nombreuses voies enzymatiques par rapport à M. tuberculosis, autre pathogène intra-cellulaire en certains points semblable à M. leprae, explique les différences observées entre les deux organismes. Enfin, l'analyse génomique du bacille lépreux permet de comprendre les bases moléculaires de sa résistance à différents antibiotiques et d'identifier des cibles potentielles pour la mise au point de nouveaux traitements.
Assuntos
Humanos , Análise de Sequência de DNA , Cosmídeos/genética , Cromossomos Bacterianos/genética , DNA Bacteriano/análise , Genoma , Hanseníase/fisiopatologia , Hanseníase/tratamento farmacológico , Mycobacterium leprae/fisiologia , Mycobacterium leprae/genética , Mycobacterium leprae/patogenicidade , PlasmídeosRESUMO
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.
Assuntos
Mapeamento Cromossômico , Leishmania major/genética , Transcrição Gênica , Animais , Sequência de Bases , Cosmídeos , DNA de Protozoário/química , DNA de Protozoário/genética , Endodesoxirribonucleases , Regulação da Expressão Gênica , Biblioteca Genômica , Dados de Sequência Molecular , Mutagênese Insercional , RNA de Protozoário/genética , Sequências Repetitivas de Ácido Nucleico , Mapeamento por Restrição , Telômero/genéticaRESUMO
In Streptomyces venezuelae, four polyketide synthase (PKS) polypeptides encoded by pikAI-pikAIV are used to generate 10 and 12-membered macrocyclic structures, narbonolide and 10-deoxymethynolide. Sequence analysis suggests these genes are translationally coupled with downstream genes, pikAV (encoding a type II thioesterase), desVIII-desVI (encoding enzymes responsible for production of the final glycosylated products pikromycin, narbomycin, methymycin and neomethymycin) and desR (a resistance gene). Type II thioesterases have been suggested to have an editing function in polyketide biosynthesis and deletion of the corresponding genes often leads to decreased levels of polyketide production. Surprisingly an in-frame deletion of 687 bp of the 843 bp pikAV ORF led to a strain SC1022 that produced normal yields of polyketide products, but only in the aglycone form. Plasmid-based expression of the desVIII-VI and desR in the SC1022 strain completely restored production of glycosylated products, despite the absence of a functional pikAV gene product. Under these conditions the PikAV TEII therefore does not play an important role in polyketide biosynthesis, and its function remains an enigma. These observations also demonstrate that the region of pikAV DNA deleted in strain SC1022 contains a transcription unit essential for expression of the des genes. A sequence alignment of PikAV with members of the highly conserved type II thioesterases revealed a short divergent region at the carboxy terminus, suggesting a region of pikAV that might contain such a transcription unit. DNA containing this region of pikAV was shown to be able to increase plasmid-based expression of both crotonyl CoA reductase gene (ccr) and the erythromycin resistance gene (ermE) in S. venezuelae.
Assuntos
Ácido Graxo Sintases/genética , Streptomyces/genética , Tioléster Hidrolases/genética , Acil-CoA Desidrogenases , Sequência de Aminoácidos , Antibacterianos/metabolismo , Proteínas de Bactérias/genética , Cosmídeos/genética , DNA Recombinante , Ácido Graxo Sintases/metabolismo , Regulação Bacteriana da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Teste de Complementação Genética , Glicosilação , Lactonas/química , Lactonas/metabolismo , Macrolídeos/metabolismo , Dados de Sequência Molecular , Mutação , Óperon , Oxirredutases/genética , Oxirredutases/metabolismo , Deleção de Sequência , Homologia de Sequência de Aminoácidos , Streptomyces/enzimologia , Streptomyces/metabolismo , Tioléster Hidrolases/metabolismo , Fatores de Tempo , Transcrição GênicaRESUMO
A cosmid library was made of the 2.7 Mb genome of the Gram-negative plant pathogenic bacterium Xylella fastidiosa and analysed by hybridisation mapping. Clones taken from the library as well as genomic restriction fragments of rarely cutting enzymes were used as probes. The latter served as a backbone for ordering the initial map contigs and thus facilitated gap closure. Also, the co-linearity of the cosmid map, and thus the eventual sequence, could be confirmed by this process. A subset of the eventual clone coverage was distributed to the Brazilian X.FASTIDIOSA: sequencing network. Data from this effort confirmed more quantitatively initial results from the hybridisation mapping that the redundancy of clone coverage ranged between 0 and 45-fold across the genome, while the average was 15-fold by experimental design. Reasons for this not unexpected fluctuation and the actual gaps are being discussed, as is the use of this effect for functional studies.
Assuntos
Biblioteca Gênica , Genoma Bacteriano , Bactérias Gram-Negativas/genética , Brasil , Cromossomos Bacterianos/genética , Cosmídeos , Hibridização de Ácido Nucleico , Plantas/microbiologiaRESUMO
Six different domains of CAG repeats from a human chromosome 12-specific cosmid library were identified, cloned, and sequenced. These CAG repeat domains were localized into the human chromosomic region 12q24.1. Five of them constitute repeat candidates for expansions in autosomal dominant neurological disorders with genetic anticipation, and they can also contribute to the chromosome walking in the human genome project.