John R. Young

Use of species-specific DNA probes for detection and identification of trypanosome infection in tsetse flies

Species- and subspecies-specific trypanosome DNA hybridization probes have been employed in the detection and identification of trypanosome infections in Glossina morsitans centralis. Several ways of sample preparation including the use of tsetse organ suspensions, proboscides and dissected midguts, as well as tsetse abdominal content touch-blots were explored. The results of hybridization of radio-isotope-labelled species-specific DNA probes to tsetse samples indicated that it was possible to detect trypanosomes in the organs where parasite development is known to characteristically occur for each subgenus. Duplicate slot-blots of samples prepared from midguts of tsetse infected with 2 strains of T. congolense and from non-infected fly controls show that it is not only possible to detect infection in tsetse but also to identify the strain of parasite present in a sample after hybridization with the DNA probes specific for each strain. The results, obtained after hybridization of sequential abdominal touch-blots from the same fly with the DNA probe specific for one strain of T. congolense, indicated that at least 8 positive signals can be observed after an overnight exposure. Because of their simplicity and potentially low cost, the techniques described here would be appealing for screening large numbers of tsetse samples from the field for the presence of any trypanosome residing in the guts or proboscis of the vector. In addition, the possibility of doing multiple touch-blots from the same fly gives the opportunity of detecting mixed trypanosome infections in the vector.

SfiI and NotI polymorphisms in Theileria stocks detected by pulsed field gel electrophoresis

DNAs of Theileria parva parva, T. p. lawrencei, T. p. bovis and Theileria mutans stocks, from Kenya, Uganda, Zanzibar and Zimbabwe were digested with either SfiI or NotI and analysed using contour-clamped homogeneous electric field (CHEF) and field-inversion gel electrophoresis (FIGE). The SfiI-digested T. parva genomic DNA resolved into approximately 30 fragments while the NotI digestion produced between 4-7 bands. The summation of the sizes of SfiI fragments gave an estimate of 9-10 X 10(6) base pairs for the size of the T. parva genome. Heterogeneity within T. p. parva Muguga, Pemba/Mnarani and Mariakani stocks was detected. All the T. parva stocks analysed showed SfiI and NotI restriction fragment length polymorphisms (RFLP). Hybridisation of 5 SfiI-digested T. parva DNAs with a Plasmodium berghei telomeric repeat probe suggested that most of the polymorphisms and heterogeneity occurred in the telomeric or sub-telomeric regions of the genome. The recognition by the Plasmodium telomeric probe of 7-8 strongly hybridising SfiI bands indicates that the T. parva genome may possess at least 4 chromosomes. The T. mutans genome was cut frequently with the above enzymes resulting in large numbers of fragments predominantly below 50 kb, thus suggesting either a much higher G + C content than T. parva or the presence of highly reiterated G + C-rich regions.

Relationship between multiple copies of a T. brucei variable surface glycoprotein gene whose expression is not controlled by duplication

Unlike many other T. brucei variable surface glycoprotein (VSG) genes, the IITat 1.3 gene is not duplicated when it is expressed. Analysis of the multiple copies of this gene present in all IITaR 1 trypanosome clones by restriction enzyme mapping and sequencing shows that the expressed copy may have arisen by duplication and transposition to a telomeric site, as is observed for those VSG genes whose expression is linked to duplication. The existence of a mechanism selecting between a number of complete telomeric VSG gene copies for expression is implied by these results. Comparisons of the nontelomeric copies of the IITat 1.3 gene are consistent with involvement of gene duplication and mutational drift in the evolution of new VSG genes.

Minichromosomal variable surface glycoprotein genes and molecular karyotypes of Trypanosoma (Nannomonas) congolense

Employing orthogonal-field-alternation gel electrophoresis (OFAGE), we have separated chromosome-sized DNA molecules from Trypanosoma (Nannomonas) congolense clones, the clones being derived from several distinct antigenic repertoires. Trypanosome clones that belong to a specific antigenic repertoire appear to have a chromosome pattern characteristic of that particular repertoire. Hybridization of the separated chromosomes with cloned DNA fragments encoding variable surface glycoproteins revealed the presence of two different T.(N.) congolense variable surface glycoprotein genes on mini-chromosomes (mc) and the modes by which these genes may be activated: one by duplicative and the other by non-duplicative activation.

Barbed Hooklets on Spines of a Brachiopod

MR. THOMAS DAVIDSON, F.R.S., describes, on p. 275, and figures, in pl. xxxiv. of the Supplement to his “Carboniferous Brachiopoda,” now on the eve of publication, some important points in the structure of Spirifera lineata, Martin, which specimens in rny collection have revealed. In this species the shell structure is minutely punctate, and the flattened spines, which are usually broken] off short, contain in their interior a double canal, that terminates upon the outer surface of the shell in a series of double pores. I have recently been fortunate enough to find a specimen from the High Blantyre limestone shales having the spines in place. It appears that these spines are provided with numerous marginal opposite booklets usually pointing towards the free end of the spine. So far as I am aware, this structure is unique amongst the brachiopods. Mr. Davidson has kindly undertaken to note this interesting fact in the explanation of the plates of his forthcoming monograph, the text having been printed off before this observation was made; but I should like to draw the attention of palæontologists to the point, as perhaps similar structures may be found in other brachiopods. The materials are in Mr. Davidsons hands for extended notice when his leisure allows him.