The establishment of genomic DNA libraries for the human malaria parasite Plasmodium falciparum and identification of individual clones by hybridisation

The DNA of Plasmodium falciparum has been purified and fragmented with the restriction endonucleases EcoRI and HindIII. The fragments have been incorporated in vitro into derivatives of bacteriophage lambda to make libraries in which most of the parasite DNA is represented. By Southern hybridisation we have been able to recover from these libraries specific clones containing (a) repetitive DNA sequences, (b) rRNA gene(s) and (c) sequences homologous to an actin gene probe. Parasite DNA from two independent sources differs markedly in the pattern of its repetitive DNA visualised by hybridisation to our repetitive clone. By contrast, the rRNA genes of the two isolates prove to be carried on identically sized fragments.     

Further analysis of intraspecific variation in Trypanosoma brucei using restriction site polymorphisms in the maxi-circle of kinetoplast DNA

We have compared the maxi-circle kinetoplast DNA of 21 Trypanosoma brucei sp. stocks by analysis of restriction sites for nine restriction endonucleases. The analysis shows most of these stocks to have a maxi-circle sequence similar to that of 11 previously analysed stocks, with a difference of less than 3% between any two stocks. However, seven stocks stand out from the rest with at least two sites lost or gained for six of the nine restriction enzymes used. These seven distinctive stocks fall into two groups with some shared and some unique polymorphisms. One group had already been designated the kiboko group on the basis of isoenzyme patterns, but the relationship between nuclear markers and maxi-circle type is less clear-cut for the other group, designated sindo. Both groups seem to be in a wild animal-tsetse fly transmission cycle, with occasional infections in domestic stock, and may be reproductively isolated from the main T. brucei sp. population. The existence of the kiboko and sindo sub-groups shows that the maxi-circle is not shielded from evolutionary change. The lack of difference observed between the maxi-circles of the majority of T. brucei sp. stocks, including the gambiense and rhodesiense variants, must therefore reflect their close homology. Two geographical trends occur in T. brucei as a whole: (a) a trend in maxi-circle size, with increasing length of the variable region from West to East Africa, and (b) a greater frequency of certain restriction enzyme polymorphisms in East African stocks as compared to West African stocks.     

Quantitation of 5-bromo-2-deoxyuridine incorporation into DNA: an enzyme immunoassay for the assessment of the lymphoid cell proliferative response

As an alternative to the measurement of radiolabeled thymidine incorporated into DNA, a method is presented in which thymidine has been replaced by its analogue, 5-bromo-2-deoxyuridine (BUdR). BUdR incorporated into DNA (BUdR-DNA) is measured by a sandwich-type enzyme immunoassay using a monoclonal anti-BUdR antibody. This method allows the quantitation of 4 ng of BUdR-DNA. Comparative experiments with myeloma cells and LPS stimulated spleen B-cells have shown that this technique is at least as sensitive as the traditional counting of [3H]thymidine.     

Tailoring and histochemical application of fluorescent homo-dimeric styryl dyes using frozen sections: from peroxidase substrates to new cytochemical probes for mast cells, keratin, cartilage and nucleic acids

Homo-dimers of styryl dyes were chemically tailored in order to become specific cytochemical probes for use in the life sciences. Histochemical applications using fixed cryotome sections are discussed. It is concluded, that homo-dimerization of specific styryl substrates of peroxidase (PO) by way of their covalent linkage, does not necessarily lead to improved detection sensitivity of endogenous and immuno-bound peroxidase (PO) activity. In general, these dimers act less specific towards PO activity than parent monomers. Synergetic interactions of the doubled basic dye compartments with cell constituents cause a pronounced staining of further targets at the cellular level. This behavior depends on the functional groups present in each dye compartment in a crucial manner. However, by way of chemical dye tailoring centering of these initially unwanted staining properties is possible leading to novel highly fluorescent stains for mast cells, nucleic acids, keratin and cartilage tissue. Structure/staining behavior-relationships of these stains are discussed.