Assignment of Borrelia burgdorferi strains G25 and VS461 to the Borrelia garinii and Borrelia afzelii genospecies, respectively: a comparison of OspA protein sequences.

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.

Molecular cloning and analysis of two tandemly linked genes for pyruvate kinase of Trypanosoma brucei.

In Trypanosoma 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 54,378 Da and 54,363 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.

Characterization of the genes for fructose-bisphosphate aldolase in Trypanosoma brucei.

In Trypanosoma brucei stock 427 the glycolytic enzyme fructose-bisphosphate aldolase is encoded by two tandemly linked genes of identical sequence. Such a tandem arrangement of aldolase genes is also present in other T. brucei stocks of unrelated origin. In stock 427 one of the allelic genes is a pseudogene, as a result of a one-nucleotide deletion. The genes code for a polypeptide of 371 amino acids, with a calculated molecular weight of 40,940. The protein that is predicted from the gene sequence has 45-48% positional identity with known aldolase sequences of other organisms. The trypanosomal protein is, however, unique in having a 10 amino-acid insertion near its N-terminus and high number of basic residues, a feature it shares with other glycolytic enzymes of T. brucei. These glycolytic enzymes have in common that they are located in microbody-like organelles, the glycosomes. We have previously proposed that the positively charged residues may be involved in the import of the proteins into the organelles.

Two tandemly linked identical genes code for the glycosomal glyceraldehyde-phosphate dehydrogenase in Trypanosoma brucei.

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.