John E. Donelson

Sequence diversity and organization of the msp gene family encoding gp63 of Leishmania chagasi

During in vitro growth Leishmania chagasi promastigotes differentially express 3 classes of RNAs encoding the major surface protease (MSP) gp63 that can be distinguished by their unique 3 untranslated regions. Here we show that the three classes (logarithmic-specific, stationary-specific and constitutively expressed) are encoded by a family of at least 4 tandem stationary genes (mspS2, mspS1, mspS3 and mspS5) followed by twelve or more logarithmic genes (mspL genes), one constitutive gene (mspC) and a final stationary gene (mspS4). Some of the stationary genes can be distinguished from each other by groups of nucleotide differences within the coding regions that result in localized amino acid differences. Northern blots confirm that RNAs from the individual stationary genes are present in stationary, but not logarithmic, phase promastigotes. Western blots using sera directed against synthetic peptides indicate that correspondingly heterogeneous gp63 proteins are expressed in L. chagasi promastigotes. A 200-bp region upstream of all three gp63 gene classes is conserved except for a variable number of 6-bp repeats. Downstream of the gp63 coding regions are highly conserved, class-specific sequences that include the 3 untranslated regions and extend past the polyadenylation site for 65 bp (mspL), 345 bp (mspC) or 2.8 kb (mspS). These sequence features flanking the msp coding regions are likely important in the growth phase-specific expression of the three gp63 RNA classes.

OvGalBP, a filarial antigen with homology to vertebrate galactoside-binding proteins

The increased incidence of allergic symptomatology and clinical complications seen in non-endemic individuals with loiasis, as compared to natives of endemic areas, is thought to reflect a heightened immune response to filarial antigens. To identify antigens involved in this hyperresponsiveness, a cDNA library constructed from adult female RNA from the related filarial parasite, Onchocerca volvulus, was screened with serum from a North American who acquired loiasis in West Africa. Sequence analysis of one of the identified clones, OvGalBP, revealed significant homology to the vertebrate S-type lectins, a family of thiol-dependent, metal-independent galactoside binding lectins, which includes an IgE-binding protein thought to be involved in IgE regulation. The 1100-bp insert of OvGalBP contains the entire protein coding region and has a 3 poly(A) tail. The two amino acid consensus sequences (WGxExR and HFNPRF) found in all of the S-type lectins are present. Purified recombinant protein expressed as a fusion with glutathione-S-transferase (OvGalBP-GST) was recognized by sera from a majority of filaria-infected patients but not by putatively immune individuals from an endemic area or by unexposed endemic and non-endemic controls. Interestingly, OvGalBP-GST specifically bound IgE (and not IgG) in a lactose-inhibitable manner, suggesting a potential role for this protein in the pathophysiology of human filarial infection.

A mitochondrial heat shock protein from Crithidia fasciculata

MCP72 is a mitochondrial hsp70 protein from the trypanosomatid Crithidia fasciculata. An MCP72 cDNA clone was isolated from a C. fasciculata cDNA library by screening with antiserum specific for the homologous protein of Trypanosoma cruzi [9]. The MCP72 cDNA encodes a polypeptide of 663 amino acids which is 84% identical to the Trypanosoma cruzi protein and 56% identical to the Escherichia coli hsp70 protein DnaK. MCP72 is less similar to other hsp70 proteins. Native MCP72 was purified to homogeneity by ATP-agarose affinity chromatography. Comparison of its N-terminal amino acid sequence with that deduced from the cDNA sequence shows that 20 amino acid residues had been cleaved from the N-terminus; this sequence probably represents a mitochondrial import signal which is cleaved during translocation into the mitochondrion. Fluorescence microscopy, using antibodies specific for MCP72, indicates that the protein is concentrated in a region of the mitochondrial matrix which surrounds the kinetoplast.

The promoter for the ribosomal RNA genes of Leishmania chagasi

A promoter for the rRNA genes of Leishmania chagasi was found to be located about 1 kb upstream of the 18S rRNA coding region and immediately downstream of 64 bp tandem repeats. Its approximate boundaries and corresponding transcription start site were determined by transient transfections and primer extension assays. This promoter for RNA polymerase I has differing activities when transfected into various Leishmania species and no activity in Trypanosoma cruzi. Its sequence has no obvious similarities with other known rRNA promoters in Trypanosomatids. Depending on the species, this promoter can be used to increase expression of a protein from a plasmid in Leishmania by as much as 45-fold over that from a plasmid lacking a promoter.

Many transcribed regions of the Onchocerca volvulus genome contain the spliced leader sequence of Caenorhabditis elegans.

Genomic DNAs of the related parasitic nematodes Onchocerca volvulus and Dirofilariae immitis, and a cDNA library of O. volvulus, were examined for the presence of the 22-nucleotide spliced leader (SL) found at the 5 ends of 10 to 15% of the mRNAs in the free-living nematode Caenorhabditis elegans. As in C. elegans, genes for the SL RNA are linked to the repetitive 5S rRNA genes of O. volvulus and D. immitis, but unlike C. elegans, they are in the same orientation as the 5S rRNA genes within the repeat unit. In O. volvulus the SL sequence is also encoded at more than 30 additional genomic locations and occurs at interior sites within many transcripts. Sequence determinations of four different cDNAs of O. volvulus, each containing an internal copy of the SL within a conserved 25mer, and one corresponding genomic DNA clone indicate that this sequence is not trans spliced onto these RNAs, but is encoded within the genes. The RNAs of two of these cDNAs appear to be developmentally regulated, since they occur in adult O. volvulus but were not detected in the infective L3 stage larvae. In contrast, actin mRNAs are present at all developmental stages, and at least one actin mRNA species contains a trans-spliced 5 SL. The internal locations of the SL in various transcripts and its perfect sequence conservation among parasitic and free-living nematodes argues that it serves specific, and perhaps multiple, functions for these organisms.

Differential expression of two mRNAs from a single gene encoding an HMG1-like DNA binding protein of African trypanosomes

We have isolated cDNA clones expressing a member of the high mobility group (HMG) protein family by screening a Trypanosoma brucei rhodesiense expression cDNA library with multimerized oligonucleotides corresponding to an octamer transcriptional regulatory sequence motif. The trypanosome DNA binding protein (TDP-1) encoded by these cDNAs contains two domains that show striking sequence similarity to the consensus sequence for HMG1-like DNA binding domains (HMG boxes). Southern blot analysis is consistent with TDP-1 being encoded by a single copy gene. The cDNA clones are derived from 2 mature mRNA species of approximately 1.6 and 2.3 kb in length that are generated by differential polyadenylation at sites 563 nucleotides and 1113 nucleotides downstream from the stop codon. Stage specific differences exist in the steady state levels of the 2 mRNAs: bloodstream parasites contain predominantly the 1.6-kb mRNA, while procyclic culture forms express predominantly the 2.3-kb mRNA.

Characterization of trypanosome protein phosphatase 1 and 2A catalytic subunits.

Oligonucleotides corresponding to highly conserved regions of mammalian protein phosphatase catalytic subunits were used in the polymerase chain reaction (PCR) to generate an amplification product from genomic DNA of Trypanosoma brucei rhodesiense. The PCR product was used to screen a T. b. rhodesiense cDNA library for cDNA clones encoding putative protein phosphatase catalytic subunits. Two cDNA clones, (TPP1A and TPP1B) representing two distinct type 1 catalytic subunit isotypes, encode 39-kDa proteins of 346 amino acids that show 66% and 40% identity, respectively, to mammalian protein phosphatase 1 and 2A catalytic subunits. Both cDNAs are derived from 2.3-kb mRNAs, and Northern blot analysis has provided indirect evidence that these mRNAs are part of the same transcription unit as mRNAs for RNA polymerase II largest subunit. Another cDNA, TPP2, represents the type 2A class of phosphatases and codes for a 34.5-kDa protein of 303 amino acids. The deduced amino acid sequence has 39% and 55% identity, respectively, to the catalytic subunits of mammalian protein phosphatase 1 and 2A. Southern and Northern blot analyses are consistent with TPP2 being encoded by a single copy gene from which is derived a mRNA of 2.5 kb. This finding constitutes the first example in eukaryotes in which a single gene encodes the type 2A class of protein phosphatases. Sera from mice immunized with TPP1A fusion protein reacted with the catalytic subunits of mammalian types 1, 2A and 2B protein phosphatases. However, antisera to TPP2 fusion protein was specific for the type 2A catalytic subunit and recognized a polypeptide of 35 kDa in a Western blot of crude trypanosomal lysate.

Generation of expressed sequence tags as physical landmarks in the genome of Trypanosoma brucei

Previous molecular genetic studies on the African trypanosome have focused on only a few genes and gene products, the majority of which are concerned with surface antigenic variation; consequently, an insignificant number of the genes of this organism have been characterized to date. In order to: (1) identify new genes and analyze their expression profile, (2) generate expressed sequence tags (ESTs) for derivation of a physical map of the trypanosome genome, and (3) make available the partial sequence information and the corresponding clones for general biomedical research on the parasite, we have performed single-pass sequencing of random, directionally cloned cDNAs from a bloodstream form Trypanosoma brucei rhodesiense library. Analysis of 2128 such ESTs sequenced so far in this study showed significant similarities [BLASTX P(n)-value < 10(-4), and a match > 10 amino acid residues] with proteins whose genes have been described in diverse organisms including man, rodents, kinetoplastids, yeasts and plants. A number of the ESTs encode homologues of proteins involved in various functions including signal reception and transduction, cell division, gene regulation, DNA repair and replication, general metabolism, and structural integrity. Although some of these genes may have been expected to be present in the African trypanosomes, the majority of them had not previously been described in these organisms. A large proportion, 768 individual ESTs (36%, representing 385 different transcripts), had a significant homology with genes described in organisms other than the African trypanosomes; however, 15% of the ESTs were from genes already described in trypanosomes. Among the ESTs analysed were 462 distinct known genes, only 77 of which have been described in T. brucei. Approximately 52% of the ESTs did not show any significant homology with the sequences in any of the public domain databases.

A comparison of trans-RNA splicing in trypanosomes and nematodes

Many aspects of the biology of kinetoplastids are unique, so it is surprising that they share with nematodes an unusual post-transcriptional process called trans-splicing. During this process, a small conserved RNA sequence is added to the 5 non-translated ends of transcribed RNAs of protein-encoding genes. Trypanosomes and nematodes are the only organisms to date in which these sequences have been described, and the biological significance of trans-splicing remains a mystery but may be of wider occurrence in invertebrates. In this review, John Donelson and Wenlin Zeng compare the process in nematodes and trypanosomes and speculate on its raison dêtre.

The actin genes of Onchocerca volvulus

The genome of Onchocerca volvulus was found to contain 2 actin gene classes (called 1 and 2) of 2 genes each. The 4 genes are located in 2 clusters (called A and B), each containing a gene class member. Five short introns of 122-207 bp occur within each gene. The sequences of the fourth intron and the 5 and 3 untranslated regions of all the 2 gene classes are completely different even though their coding regions share 95% identity. Mature transcripts from the actin genes have the nematode spliced leader (SL) at their 5 ends. One actin cDNA was found to be derived from an actin pre-mRNA which locks both the 5 introns and the 5 SL, suggesting that in at least some transcripts cis-splicing is completed before trans-splicing occurs.