954 resultados para Lambda probe


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We describe the main scientific goals to be addressed by future in situ exploration of Saturn.

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M.J. L-u.

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Israel Kley

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David ben Mose Fränkel

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The number of immunoglobulin G constant heavy chain genes (cgamma genes) varies broadly among mammalian species, reflecting structural and functional differences between expressed immunoglobulin G (IgG) isotypes and allotypes. Up to now equine IgG isotypes have been defined only at the biochemical and serological level. It is still not clear how many IgG isotypes exist in horses and whether there are any allotypes. Here, we describe the isolation and characterisation of equine cgamma genes. An equine genomic lambda phage library was screened with a human cgamma4 probe. Cross-hybridising equine cgamma sequences were cloned twice and characterised by restriction mapping with the human cgamma4 and a murine sgamma1 probe. Genomic equine DNA probes for both, cgamma genes and corresponding switch regions (sgamma), were isolated and used for a more detailed BamHI restriction analysis, comparing genomic DNA of various horses. This analysis reveals the existence of at least five, or probably six cgamma genes in the equine haploid genome. Beside the porcine system, this is the highest number of cgamma genes described for any mammalian species. Moreover, for two of these cgamma genes, BamHI restriction fragment length polymorphism became evident.

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Degenerate oligonucleotide primers derived from conserved cysteine protease sequences were used in the reverse transcription polymerase chain reaction to amplify seven different cysteine protease cDNA clones, Fcp1-7, from RNA isolated from adult Fasciola hepatica. Five of the amplified F. hepatica sequences showed homology to the cathepsin L type and two were more related to the cathepsin B type. Southern blot analysis suggests that some members of this protease gene family are present in multiple copies. Northern blot analysis revealed differences in the levels of steady state mRNA expression for some of these proteases. The 5' and the 3' regions of Fcp1 were amplified using the rapid amplification of cDNA ends PCR protocol (RACE-PCR) and an additional clone was obtained by screening a lambda gt10 cDNA library using Fcp1 as a probe. The Fcp1 cDNA fragment was also subcloned in the expression vector pGEX and expressed as a glutathione-S-transferase (GST) fusion protein in Escherichia coli. Antibodies, raised in rabbits against the GST:Fcp1 fusion protein, were used in western blot analysis to examine expression in different life-cycle stages of F. hepatica. In extracts from adult and immature parasites, the immune serum recognised predominantly two proteins of 30 kDa and 38 kDa. In other parasite stages, proteins of different molecular weight were recognised by the anti-GST:Fcp1 antiserum, indicating stage-specific gene expression or processing of Fcp1. In gelatine substrate gel analysis, strong proteolytic activity could be detected at 30 kDa, but not at 38 kDa, suggesting that the 30 kDa protein represents the mature enzyme and the 38 kDa protein the proenzyme.

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Repetitive DNA sequences present in the genome of Dicrocoelium dendriticum were identified by hybridization of genomic DNA that had been digested with different restriction enzymes with 32P-labeled genomic D. dendriticum DNA. DNA fragments containing repetitive sequences were isolated from PstI-digested D. dendriticum DNA and were subcloned into a plasmid vector. Plasmids containing repetitive sequences were identified by colony hybridization. One of these plasmids, designated Ddr-IV, was isolated and used as a probe in further studies. Ddr-IV is specific for D. dendriticum since it does not hybridize to DNA isolated from other trematodes. In addition, Ddr-IV was capable of detecting D. dendriticum metacercariae in ants (Formica cunicularia, F. rufibarbis, and Lasius sp.), which act as second intermediate hosts in the parasite's life cycle. Since metacercariae constitute the infectious stage of the parasite for grazing animals, Ddr-IV will provide a useful tool for epidemiology studies of dicrocoeliosis.

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M. E. Stern

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Abraham Basch