Detection of genetic polymorphism among and within Echinococcus granulosus strains by heteroduplex analysis of a microsatellite from the U1 snRNA genes

Polymerase chain reaction of a pentanucleotide microsatellite in the U1 snRNA gene complex generated a multiple band pattern due to the priming of paralogous sequences. Denaturation and slow renaturation of polymerase chain reaction products allow the formation of heteroduplex DNA that can be detected by its differential mobility in polyacrylamide gel electrophoresis. Heteroduplex analysis was used to determine if the U1 snRNA microsatellite could be a useful genetic marker in Echinococcus granulosus. A U1 snRNA microsatellite fragment from E. granulosus was isolated and characterized by Southern blot and sequencing. Four E. granulosus strains were analyzed: sheep, Tasmanian sheep, cattle, and camel strains. The former two showed polymorphism and shared three of the six patterns found for sheep strain. The cattle strain displayed two patterns, and the camel strain was monomorphic. The electrophoretic profiles were used for statistical analysis in order to determine genetic distance and the relationship among strains. Heteroduplex analysis can be helpful in genotyping E. granulosus strains and is useful in detecting polymorphism within strains.

Cloning and characterization of Echinococcus granulosus (Cestode) EgactI and EgactII actin gene promoters and their functional analysis in the NIH3T3 mouse cell line

We report here for the first time the structure and function of a promoter from a cestode. The ability of DNA fragments respectively encompassing the 935-bp and 524-bp regions upstream from the ATG codon from the EgactI and EgactII actin genes of Echinococcus granulosus to promote transcription was studied in the NIH3T3 mouse cell line. The results of transfection assays showed that both regions have strong promoter activity in these cells. The fragments were tested in both orientations and the 524-bp fragment of EgactII presented a bidirectional promoter activity. Deletion analysis of EgactI and EgactII promoters indicated the presence of regulatory regions containing putative silencer elements. These results indicate that both EgactI and EgactII promoters are functional and that the preliminary functional evaluation of E. granulosus and possibly of other cestode promoters can be performed in heterologous cell lines

Selection, recombination and history in a parasitic flatworm (Echinococcus) inferred from nucleotide sequences

Three species of flatworms from the genus Echinococcus (E. granulosus, E. multilocularis and E. vogeli) and four strains of E. granulosus (cattle, horse, pig and sheep strains) were analysed by the PCR-SSCP method followed by sequencing, using as targets two non-coding and two coding (one nuclear and one mitochondrial) genomic regions. The sequencing data was used to evaluate hypothesis about the parasite breeding system and the causes of genetic diversification. The calculated recombination parameters suggested that cross-fertilisation was rare in the history of the group. However, the relative rates of substitution in the coding sequences showed that positive selection (instead of purifying selection) drove the evolution of an elastase and neutrophil chemotaxis inhibitor gene (AgB/1). The phylogenetic analysis revealed several ambiguities, indicationg that the taxonomic status of the E. granulosus horse strain should be revised.

Expression of the VP3-VP1 sequence of foot-and-mouth disease virus in Escherichia coli

1. cDNA recombinants containing the VP3 and VP1 sequences of foot-and-mouth disease virus were isolated and the VP3-VP1 sequence was reconstructed. 2. The reconstructed VP3-VP1 sequence was subcloned into expression vector pEX31b and a fusion protein of about 62,000 Da was expressed. 3. When injected into mice, the fusion protein was able to elicit the production of antibodies that recognized viral VP1 and VP3. 4. Antibodies present in sera from mice immunized with VP3-VP1 protein did not neutralize the foot-and-mouth disease virus in vitro

The nifHDK operon in the free-living nitrogen-fixin bacteria azospirillum brasiliense sequentially comprises genes H, D, K, an 353 BP orf and gene Y

The complete nucleotide sequence of the nitrogenase structural genes form Azospirillum brasilense was determined. Two additional open reading frames of 353 and 683 base pairs were detected downstream of the nifK gene. Structures resembling the consensus nif promoter and NifA-binding motif were found only upstream from the nifH region and an inverted repeat structure locate downstream of the nifY gene may be a potential stem-and-loop transcriptional terminator. The nif structural genes of Azospirillum brasiliense are transcribed as a transcription unit and organized as nifHDK orf1 Y, NifH, NifD and NifK polypeptides share significant sequence identies when compared to nif structural gene products from other organisms. The three polypeptides are characterized by the presence of highly conserved cysteine residues which may play a role in binding the iron-sulfur cluster

Molecular cloning and characterization of the ribosomal RNA genes of the cestode Echinococcus granulosus

The analysis of total protoscolex DNA and some rDNA recombinats of Echinococcus granulosus by restriction endonuclease mapping and hybridization to rDNA probes indicated the complex organization of the ribosomal RNA genes and that some repeat units are larger than 15 kb. The nontranscribed spacer can be up to 13 kb in length in some repeat units. Restriction site polymorphisms was detected mainly in the nontranscribed spacer regions although some polymorphisms was also observed in the 28S rRNA coding region. On the basis of Southern blot hybridization using EcoRi-digested genomic DNA, we conclude that the repeat units containing an extra EcoRI site are present almost in the same proportion as the repeat units without the extra EcoRi site in the 28S rRNA coding region

Construction of a gene library from Azospirillum brasilense and characterization of a recombinant containing the nif structural genes

1.We have constructed a gene library, from Azospirillum brasilense using the vector EMBL4. 2. A recombinant containing the nif structural genes from A. brasilense was isolated and characterized. This recombinant contains a DNA insert of about 15 kilobases (KB) which gives rise to five fragments after cleavage with EcoRI. Only one of the DNA fragments (6.5 Kb) hybridized to the nifHDK genes of Klebsiella pneumoniae. 3 The organization of the nif genes in this DNA fragment was determined using different DNA segments containing the nifH, nifK or nifD genes of K. pneumoniae as probes