Wallace R. Fish

Three genes and two isozymes: Gene conversion and the compartmentalization and expression of the phosphoglycerate kinases of Trypanosoma (Nannomonas) congolense

The glycosome, a microbody organelle found only in kinetoplastid protozoa, compartmentalizes the first six enzymes of glycolysis. In order to better understand the regulation and targeting of glycolytic enzymes in trypanosomes, we have cloned and analyzed the three genes of the phosphoglycerate kinase (PGK) complex of Trypanosoma (Nannomonas) congolense. The organization of the genes within the complex is similar to that of Trypanosoma brucei brucei. The nucleotide and amino-acid sequences, including those of the novel high-molecular-weight 56PGK, show substantial cross-species similarity. However, the two downstream genes, c1PGK and c2PGK, encode identical isozymes in T. congolense, while they encode distinct glycosomal and cytoplasmic isozymes in T. brucei. Western analysis also indicated that there are only two isozymes in T. congolense and that these are constitutively expressed. Differential digitonin solubilization of the trypanosomes indicated that 56PGK is primarily localized to the glycosome, as expected, and that c1/c2PGK is cytoplasmic. Northern analysis demonstrates that while 56PGK is constitutively expressed, c1PGK and c2PGK mRNAs are differentially expressed in the T. congolense developmental stages. This work demonstrates that T. congolense has only one PGK isozyme, 56PGK, that is predominantly localized in glycosomes.

DEVELOPMENTAL REGULATION OF RNA EDITING AND POLYADENYLATION IN FOUR LIFE CYCLE STAGES OF TRYPANOSOMA CONGOLENSE

The accumulation of many edited mRNAs is developmentally regulated in a transcript-specific fashion in Trypanosoma brucei. In addition, these transcripts are frequently present in two size classes which differ substantially in the lengths of their poly(A) tails, and poly(A) tail length is also developmentally regulated. Previously, these phenomena have only been studied in the mammalian bloodstream and insect procyclic forms (BF and PF, respectively) of T. brucei. In this paper, we examine developmental regulation of edited RNA abundance and poly(A) tail length of 3 mitochondrially encoded RNAs in mammalian BF and 3 insect stages (PF, epimastigotes, and metacyclics) of T. congolense. T. congolense BF and PF are similar, but not identical, to these stages of T. brucei with regard to edited RNA accumulation and poly(A) tail length. At the level of edited RNA, both epimastigotes and metacyclic stage parasites appear to be pre-adapted for the respiratory mechanisms of BF but not yet down-regulated from the cytochrome-based respiration of PF since edited RNAs encoding NADH dehydrogenase components are up-regulated and edited CYb RNA is abundant in these stages. Poly(A) tail lengths of mitochondrial mRNAs appear to be regulated independently of edited RNA abundance. These results indicate that multiple mechanisms for regulation of mitochondrial gene expression are active throughout the trypanosome life cycle.

Primary structure and partial characterization of a life-cycle-regulated cysteine protease from Trypanosoma (Nannomonas) congolense.

Trypanosoma (Nannomonas) congolense is an important pathogenic parasite of domestic livestock in Africa. We have cloned a cDNA encoding a prepro-cysteine protease of this protozoan, the sequence of which indicates it is an early mRNA processing intermediate. Northern analysis demonstrates a life-cycle-stage specificity similar to previously described enzymatic data. The deduced amino-acid sequence shows extensive similarity to cysteine proteases of other parasitic protozoa, as well as papain and cathepsin L. As with other African trypanosomes, a poly-proline tract connects the catalytic domain with an unusual C-terminal extension.

The cyclopropane fatty acid of trypanosomatids

Cis-9,10-Methyleneoctadecanoic acid, one of a group of cyclopropane fatty acids commonly found in bacteria but not in eukaryotic cells, has been identified in the phosphatidylethanolamines of 27 isolates representing 5 genera of trypanosomatid flagellates (Crithidia, Leptomonas, Herpetomonas, Phytomonas, Leishmania). Its presence did not appear to be associated with endosymbiotic or other microbiol associates. It was absent from 12 isolates of the genera Blastocrithidia, Endotrypanum and Trypanosoma.

Some Phytomonas and Herpetomonas species form unique iso-branched polyunsaturated fatty acids.

Four trypanosomatid flagellates of the genera Phytomonas and Herpetomonas have been found to carry out the de novo biosynthesis of a variety of iso-branched, C18, C20 and C22, polyunsaturated fatty acids, with 2-5 methylene-interrupted double bonds, which have not been described heretofore from natural materials; iso-C18 delta 6,9, iso-C18 delta 9,12, iso-C20 delta 8,11,14, iso-C 20 delta 5,8,11,14, iso-C22 delta 4,7,10,13,16. Identifications were based upon combinations of chromatographic, chemical degradative, mass spectrometric and proton nuclear magnetic resonance spectrometric techniques. Under appropriate culture conditions, 85% of the total fatty acids of the organisms were branched. The subject trypanosomatids are recommended as model organisms with which to investigate influences of the physical properties of phospholipid fatty acyl groups on eukaryotic cell membrane functions.

Biosynthesis of the novel fatty acid, 17-methyl-cis-9,10-methyleneoctadecanoic acid, by the parasitic protozoan, Herpetomonas megaseliae

Herpetomonas megaseliae, a flagellate protozoan parasite of the gut of a dipteran,Megaselia scalaris, is shown by chromatographic, spectrometric and radiotracer methods to synthesize de novo aniso-branched chain cyclopropane fatty acid, 17-methyl-cis-9,10-methyleneoctadecanoic acid.