Trypanosoma brucei contains two isoenzymes for glyceraldehyde‐phosphate dehydrogenase (GAPDH); one enzyme resides in a microbody‐like organelle, the glycosome, the other one is found in the cytosol. We show here that the glycosomal enzyme is encoded by two tandemly linked genes of identical sequence. These genes code for a protein of 358 amino acids, with a mol. wt of 38.9 kd. This is considerably larger than all other GAPDH proteins studied so far, including the enzyme that is located in the cytosol of the trypanosome. The glycosomal enzyme shows 52‐57% homology with known sequences of GAPDH proteins from 10 other organisms, both prokaryotes and eukaryotes. The residues that are involved in NAD+ binding, catalysis and subunit contacts are well conserved between all these GAPDH molecules, including the trypanosomal one. However, the glycosomal protein of T. brucei has some distinct features. Firstly, it contains a number of insertions, 1‐8 amino acids long, which are responsible for the high mol. wt of the protein. Secondly, an unusually high number of positively charged amino acids confer a high isoelectric point (pI 9.3) to the protein. Part of the additional basic residues are present in the insertions. We discuss the genomic organization of the genes for the glycosomal GAPDH and the possibility that the particular features of the protein are involved in its transfer from the cytoplasm, where it is synthesized, into the glycosome.

Rotavirus infections can be diagnosed in stool samples by serological and molecular methods. We developed a novel reverse transcriptase PCR (RT-PCR) method for the amplification of rotavirus RNA and a reverse hybridization assay on a strip to detect amplimers and identify the specific G and P genotypes present in human stool specimens. An additional aim was to permit specific identification of the rotavirus G1P[8] strain, used in the Rotarix vaccine. Novel broad-spectrum PCR primers were developed for both VP4 and VP7, permitting the amplification of a wide range of rotavirus genotypes. Primer sets comprise mixtures of defined primer sequences. For the identification of G and P genotypes, two reverse hybridization strip assays were developed. Both the VP4 and the VP7 strip contain universal probes for the detection of VP4 and VP7 sequences, irrespective of the G or P genotype. The VP4 strip contains type-specific probes for P[4], P[6], P[8], P[9], and P[10]. The VP7 strip contains type-specific probes for G1, G2, G3, G4, G5, G6, G8, and G9. In addition, probes to distinguish between wild-type G1 and G1 vaccine strain sequences were present. Testing by analysis of multiple reference strains confirmed that both RT-PCR methods allowed the detection of a broad spectrum of genotypes. RT-PCR for VP7 was more sensitive than RT-PCR for VP4, but all samples identified as positive for rotavirus antigen by an enzyme-linked immunosorbent assay (ELISA) were also positive for both VP4 and VP7. The high specificity of the reverse hybridization method was confirmed by sequence analysis as well as by type-specific PCR, and the vaccine strain could also be specifically identified. The reverse hybridization method permits accurate identification of mixed infections with different genotypes. Rotavirus genotypes for which no type-specific probes were present on the strip were adequately identified by the universal detection probes. The assay was formally validated by analyses of specificity, sensitivity, precision, accuracy, and robustness. In a panel of 149 ELISA-positive stool samples, comparison with conventional type-specific RT-PCR methods revealed the superiority of the novel method, mainly in cases of mixed rotavirus infections. This novel method permits highly accurate detection and identification of human rotavirus infections in stool samples. This validated assay could be useful for large-scale epidemiological and clinical trials.

In Trypunosomu brucei (stock 427) genes encoding the glycolytic enzyme pyruvate kinase are present on two homologous chromosomes. We have cloned and characterized one of the alleles. Two large, tandemly arranged open reading frames were found, each coding for a pyruvate kinase polypeptide of 498 amino acids. The gene sequences differ at 15 positions, resulting in five amino acid substitutions. The calculated molecular masses of the polypeptides are 54378 Da and 54363 Da. These values are somewhat smaller than those reported for the subunit molecular mass of the purified protein, which is 57 -59 kDa. However, in vitro translation of the DNA region corresponding to the open reading frame, and translation of the RNA in a wheat-germ lysate, yielded a product that comigrated exactly with the native polypeptide in SDS/PAGE.The overall identity between the sequences of the trypanosomal enzyme and the enzymes from other sources is 41 -51%. The conserved residues are not equally distributed over the polypeptide. The primary structure of domains A and, to a lesser extent, B, which constitute the active site, are rather well conserved. In contrast, the sequence of domain C, which supposedly is involved in the regulation of the enzyme activity, is much more variable.The cytosolically located pyruvate kinase of T. brucei lacks the specific features found in the majority of the glycolytic enzymes of this organism that are sequestered in a microbody-like organelle, the glycosome. It has neither a relatively high subunit molecular mass, due to unique insertions or terminal extensions, nor a high excess of positively charged amino acids. The polypeptide is shorter than that of most other pyruvate kinases and the calculated net charge is only + 3.

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The nucleotide sequence encoding the Outer Surface Protein A (OspA) from two Borrelia burgdorferi sensu lato isolates, G25 and VS461, has been determined. On the basis of a phylogenetic analysis, strains G25 and VS461 were respectively assigned to the B. garinii and B. afzelii genospecies. Comparative analysis of OspA proteins from 26 different B. burgdorferi sensu lato strains involved in Lyme disease indicated a higher heterogeneity in the B. garinii genospecies than in the two other genospecies, B. burgdorferi sensu stricto and B. afzelii.