The sequence of 1000 nucleotides at the 3' end of tobacco mosaic virus RNA has been determined. The sequence contains the entire coat protein cistron as well as regions to its left and right. Sequence characterization was by conventional methods for use with uniformly 32P labeled RNA complemented by newer methods for in vitro 5' and 3' 32P end-labeling of RNA and its subsequent rapid analysis. The noncoding region separating the coat protein cistron from the 3' terminus is 204 residues long and may be folded into a clover-leaf-type secondary structure. The distribution of termination codons to the left of the coat protein cistron suggests that the end of the adjacent cistron is separated from the beginning of the coat protein cistron by only two nucleotides. The subgenomic viral coat protein mRNA was isolated from infected tissue and shown to be capped. The nontranslated sequence separating the cap from the AUG initiation codon is 9 residues long and thus overlaps a portion of the adjacent cistron on the genome RNA.

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.

SUMMARYTwenty-seven stocks of Nannomonas trypanosomes isolated from livestock in 1982 on a ranch at Kilifi on the Kenyan coast were characterized by isoenzyme electrophoresis and by the abilities of the parasite's DNA to hybridize to two repetitive sequence DNA probes. All the Kilifi stocks which were examined had isoenzyme patterns which were markedly different from the 75 patterns previously described from 78 stocks of Trypanosoma congolense. On average only 15% of the enzyme bands present in the Kilifi stocks were present in those stocks of T. congolense which had previously been surveyed for isoenzymes. The DNA from all the Kilifi stocks which had been examined for isoenzymes hybridized with only the repetitive sequence probe isolated from a clone of a Kilifi stock. In contrast, the DNA from all 27 Kilifi stocks failed to hybridize with a repetitive sequence probe isolated from a clone from a different stock of T. congolense. Thus, the trypanosomes in all the Kilifi stocks examined were both phenotypically and genotypically discrete. These genetically discrete trypanosomes have also been detected in 2 stocks isolated from livestock from another location on the Kenyan coast. The results show that there is a wide range of genetic heterogeneity within the trypanosomes currently classified as T. congolense. We suggest that the limits of this genetic heterogeneity could represent incipient speciation.