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Biomedical subjects

J E Donelson

Publications and source records attributed to J E Donelson.

At least 55 records · Page 3Linked to original sources
PubMed1995

cis-splicing and polyadenylation of actin RNA can precede 5' trans-splicing in nematodes.

In nematodes many precursor RNAs undergo both cis-splicing (intron removal) and trans-splicing (spliced leader addition) via molecular mechanisms thought to be very similar. We examined the temporal order of trans- and cis-splicing by analyzing cDNAs of partially processed transcripts from an actin gene of the parasitic nematode, Onchocerca volvulus, which has five short introns. We found that in this model gene system trans-splicing can follow cis-splicing and polyadenylation, and we obtained no evidence for alternative pathways of splicing. This result is in contrast to the tubulin genes of African trypanosomes where trans-splicing precedes polyadenylation (1).

Actins↗
PubMed1995

Intergenic regions between tandem gp63 genes influence the differential expression of gp63 RNAs in Leishmania chagasi promastigotes.

The major surface protease, gp63, of Leishmania chagasi is encoded by 18 or more tandem msp genes that can be grouped into three classes on the basis of their unique 3'-untranslated sequences (3'-UTRs) and their differential expression. RNAs from the mspLs occur predominantly during the logarithmic phase of promastigote growth in vitro, RNAs from the mspSs are present mainly in stationary phase, and RNAs from mspCs occur throughout growth in culture. All three classes of gp63 genes are constitutively transcribed during all growth phases, indicating that their expression is post-transcriptionally regulated. Chimeric plasmids containing the three different 3'-UTRs and downstream intergenic regions (IRs) fused downstream of the beta-galactosidase (beta-gal) coding region were transfected into L. chagasi, and their effects on beta-gal RNA processing and enzymatic activity were examined. The presence of the 3'-UTRs by themselves had no substantive effect on beta-gal expression. However, the 3'-UTR from a mspS plus its IR resulted in about 20-fold more beta-gal activity and RNA in stationary phase relative to logarithmic phase cells. In contrast, the 3'-UTRs plus IRs of mspL and mspC had either no or little effect, respectively, on beta-gal expression. Thus, differential expression of the mspLs and mspSs is post-transcriptionally controlled by different mechanisms.

Animals↗
PubMed1995

Developmentally regulated expression of a novel 59-kDa product of the major surface protease (Msp or gp63) gene family of Leishmania chagasi.

All species of Leishmania express a major surface protease (Msp or gp63) that facilitates the interactions of the parasite with its environment at several steps in its life cycle. The msp gene family in Leishmania chagasi contains three classes of genes whose mRNAs are differentially expressed during parasite growth. Logarithmic phase (low infectivity) promastigotes express only 63-kDa versions of Msp, whereas stationary phase (high infectivity) promastigotes express both 63- and 59-kDa Msps. The different migrations of the 59- and 63-kDa proteins on acrylamide gels are not due to differences in N-linked glycosylation or the membrane anchor. Plasmid transfections of Leishmania demonstrate that mspS2 of the stationary gene class encodes a 59-kDa protein. Expression of the 59-kDa protein in stationary phase promastigotes ceases after about 12 weeks of in vitro cultivation when the parasites become attenuated. Attenuated parasites can be stimulated to re-express the 59-kDa Msp by passage through mice followed by several in vitro passages of recovered promastigotes. Amastigotes express yet another subset of Msp proteins. Thus, the 59-kDa product of mspS2 is expressed only in stationary phase promastigotes and only after recent exposure to environmental changes encountered in the mammalian host cell.

Amino Acid Sequence↗
PubMed1995

Trypanosoma cruzi: interruption of both alleles of a gene encoding a protein containing 14-amino-acid repeats by targeted insertion of NEOr and HYGr.

In Trypanosoma cruzi approximately 90% of the 121- and 176-kDa cytoskeletal proteins encoded by the two alleles of the TCR27 gene is composed of 14-amino-acid repeats. To gain insight into the function of the TCR27 proteins we replaced the corresponding regions of 42-nucleotide repeats in the two alleles with the NEOr and HYGr genes. Analyses of DNAs and RNAs from four clones resistant to both G418, a neomycin analogue, and hygromycin showed that in both cases the repetitive regions had in fact been deleted. In addition, the absence of expression of the 14-amino-acid repeats was confirmed in Western blots. In axenic cultures growth rates of the morphologically unchanged, doubly resistant organisms were not different from those of wild-type parasites. However, the doubly resistant organisms proliferated more slowly in cultured mammalian cells than did wild-type parasites. These findings indicate that the absence of the TCR27 repetitive regions is detrimental, but not fatal, to the parasites.

Alleles↗
PubMed1995

The putative promoter for a metacyclic VSG gene in African trypanosomes.

During their metacyclic developmental stage, African trypanosomes are coated with one of 12-15 variant surface glycoproteins (VSGs) that define different metacyclic variant antigen types (MVATs). The MVAT VSG genes are located near telomeres of large chromosomes and are expressed without rearrangement in the metacyclic stage. We have cloned and examined the telomere-linked MVAT5 VSG gene and its upstream expression site associated gene (ESAG I) which are separated by 4.5 kb. Within this 4.5-kb intergenic region is an 87-bp sequence that serves as a strong promoter for a luciferase reporter gene in transient transfection assays. This 87-bp sequence is similar, but not identical, to the promoter for another MVAT VSG gene. UV irradiation experiments were used to detect RNA synthesis from this MVAT5 promoter in bloodstream trypanosomes expressing an unrelated VSG. We propose that this sequence is a specific promoter for the MVAT5 VSG mRNA that occurs in about 10% of the trypanosome population during the metacyclic stage of the parasites' life cycle.

Animals↗
PubMed1995

cDNA expressed sequence tags of Trypanosoma brucei rhodesiense provide new insights into the biology of the parasite.

A total of 518 expressed sequence tags (ESTs) have been generated from clones randomly selected from a cDNA library and a spliced leader sub-library of a Trypanosoma brucei rhodesiense bloodstream clone. 205 (39%) of the clones were identified based on matches to 113 unique genes in the public databases. Of these, 71 cDNAs display significant similarities to genes in unrelated organisms encoding metabolic enzymes, signal transduction proteins, transcription factors, ribosomal proteins, histones, a proliferation-associated protein and thimet oligopeptidase, among others. 313 of the cDNAs are not related to any other sequences in the databases. These cDNA ESTs provide new avenues of research for exploring both the novel trypanosome-specific genes and the genome organization of this parasite, as well as a resource for identifying trypanosome homologs to genes expressed in other organisms.

Amino Acid Sequence↗
PubMed1995

A recombinant Leishmania chagasi antigen that stimulates cellular immune responses in infected mice.

Cellular immune mechanisms resulting in gamma interferon production are critical for protection against visceral leishmaniasis. Antigens stimulating T-cell responses are likely present in the intracellular amastigote form of the parasite, since this is the form found in a mammalian host. To identify T-cell antigens of Leishmania chagasi, the parasite causing South American visceral leishmaniasis, we used a double antibody-T-cell technique to screen an amastigote cDNA library. One cDNA selected (Lcr1) encodes an antigen that stimulated proliferation of splenic T lymphocytes from infected mice that were either resistant (C3H.HeJ) or susceptible (BALB/c) to L. chagasi infection. The Lcr1 cDNA contains four highly divergent 201-bp repeats homologous to the 204-bp repeat of a Trypanosoma cruzi flagellar antigen gene. Results are consistent with a single copy of the Lcr1 gene producing an mRNA of > 10 kb and a protein of > 200 kDa. Recombinant Lcr1, cloned adjacent to polyhistidine and purified on a nickel affinity column, stimulated gamma interferon but not interleukin-4 (IL-4), IL-5, or IL-10 secretion by T-cell-enriched splenocytes from either susceptible or resistant mice during L. chagasi infection. Immunization with Lcr1 partially protected BALB/c mice against challenge with L. chagasi, indicating the utility of the double screening approach in selecting relevant T-cell antigens.

Amino Acid Sequence↗
PubMed1995

Repetitive protein antigens of Trypanosoma cruzi have diverse intracellular locations.

We previously identified by immunoscreening Trypanosoma cruzi cDNA libraries a group of proteins containing long stretches of tandem repeats. The goal of the current project was to gain insight into the functions of these proteins through ultrastructural analyses consisting of western blotting and electron microscopic localization studies. By comparing western blots of total parasite lysate and different fractions of T. cruzi, we found that 3 of the repetitive antigens are exclusively associated with the parasite membrane, or cytoskeleton, or both. One of the 4 repetitive antigens studied has some association with the membrane or cytoskeleton but also appears to be free in the cytosol. In immunoelectron microscopic studies, the 4 repetitive antigens were detected in different intracellular locations. One of the proteins is located between the flagellum and parasite body, the second has a nuclear distribution, the third is associated with the cell membrane, and the fourth is dispersed throughout the cytoskeletal network. These findings suggest that despite the general structure similarities of these repetitive proteins, they may serve different cellular functions.

Amino Acid Sequence↗
PubMed1994

B lymphocytes of mice display an aberrant activation phenotype and are cell cycle arrested in G0/G1A during acute infection with Trypanosoma brucei.

Humans and domestic animals with African trypanosomiasis exhibit abnormalities of immune function characterized by polyclonal lymphocyte activation and, paradoxically, progressive immunodeficiency. Mice infected with Trypanosoma brucei clone IaTat1.2 develop a fulminant parasitemia by day 5 of infection. B lymphocytes isolated from spleens of infected mice display an aberrant activation phenotype manifested by decreased CD23 and surface IgM, increased CD69 and L-selectin, and normal levels of surface IgD, MHC class II, CD32, and transferrin receptor. Kinetic analyses showed that CD23 and surface IgM were continuously down-regulated from day 2 through day 5, whereas MHC class II was elevated on days 2 and 3, but returned to normal levels by day 5, suggesting that CD23 and MHC class II are independently regulated in T. brucei infection. The aberrant activation phenotype of B cells responding in vivo to T. brucei was accompanied by impaired responsiveness of these B cells to mitogenic stimulation in vitro. The pathologic phenotypic and functional properties of B lymphocytes from T. brucei-infected mice can be partially accounted for by the virtual total arrest of B cells in G0/G1A of the cell cycle. The B lymphocyte alterations observed in the present studies provide new insight into the immunopathology of African trypanosomiasis. We propose that terminal infection with T. brucei induces early activation events in host B lymphocytes, but that the activation response becomes aberrant by the development of what seems to be a total block in cell cycle progression.

Animals↗
PubMed1994

A differentially expressed gene family encoding "amastin," a surface protein of Trypanosoma cruzi amastigotes.

A new family of closely related glycoproteins, collectively called amastins, has been found on the surface of the amastigote form of Trypanosoma cruzi. The gene family encoding these amastigote-specific proteins was identified by differentially screening an amastigote cDNA library with reverse transcribed poly(A)+ RNA from amastigote and epimastigote stages of the parasite. Amastins are encoded by eight or more tandem genes, at least five of which are distinguished by nucleotide point changes. The 1.4-kilobase amastin mRNAs are 50 times more abundant in amastigotes than in epimastigotes or trypomastigotes. The amastin genes are transcribed to an equal extent in both amastigotes and epimastigotes, indicating that the stage-specific amastin mRNA levels are determined by a post-transcriptional mechanism. Sequence determination of full-length cDNAs reveals an open reading frame encoding 174 amino acids and a 700-base pair 3'-untranslated region. Nascent amastins contain four distinct hydrophobic regions of 20-30 amino acids each, 2 at internal locations and 1 each at the N and C termini.

Amino Acid Sequence↗
PubMed1994

Molecular and biochemical comparison of the 70-kDa heat shock proteins of Trypanosoma cruzi.

An analysis of the genetic organization, regulated expression and biochemical properties of the cytoplasmic/nuclear (hsp70) and mitochondrial (mtp70) 70-kDa heat shock proteins of Trypanosoma cruzi is presented. The two proteins are encoded by tandemly arranged gene families that are located on different chromosomes. Both are mildly heat-inducible but have different optimal temperatures for expression. During the switch from proliferation to differentiation that occurs during the growth of T. cruzi in culture, the hsp70 level decreases dramatically while the mtp70 level falls only slightly. The subcellular locations of the two proteins differ during heat shock. While mtp70 remains associated with the kinetoplast at all temperatures, hsp70 becomes more concentrated in the nucleus at higher temperatures. Biochemical analysis of hsp70 and mtp70 revealed both to be potent ATPases. Each protein binds ATP with a Km of about 70 microM and hydrolyzes ATP with a kcat of about 100 min-1, 100 times greater than the kcat of human hsp70. The high ATPase activities of hsp70 and mtp70 are further stimulated by incubation with peptides, suggesting that these trypanosome heat shock proteins have protein chaperone activity. Finally, mtp70, but not hsp70, was found to possess autophosphorylation activity in vitro, a property that it shares with prokaryotic hsp70. These findings demonstrate unique cellular and biochemical characteristics of T. cruzi mtp70 and hsp70 that suggest that they play distinct physiologic roles in the biology of the cell.

Adenosine Triphosphatases↗
PubMed1994

Transcription and editing of cytochrome oxidase II RNAs in Trypanosoma cruzi.

The cytochrome oxidase subunit II (COII) gene is one of the maxicircle cryptogenes of kinetoplastids whose primary transcripts are sometimes modified by RNA editing to produce mature mRNAs. We determined the sequence of the COII gene in three strains of Trypanosoma cruzi (Y, Corpus Christi, and Tulahuén) and examined its developmental expression. Comparison of the RNA and DNA sequences encoding COII indicated that in the three strains of T. cruzi, four uridines are inserted in the pre-mRNA at the same positions as they are in the COII pre-mRNAs of Trypanosoma brucei, Leishmania tarentolae, and Crithidia fasciculata. The putative guide RNA (gRNA) sequence that serves as a template for the four uridine insertions is located in the 3'-untranslated region of the T. cruzi COII mRNA. Analysis of editing intermediates demonstrates that the COII gRNA remains attached to the pre-mRNA while participating in the formation of chimeric RNAs. Northern blots used to investigate stage-specific expression of the COII gene revealed RNAs of 800 and 900 nucleotides, similar in size to those present in T. brucei. In contrast to the differential expression observed in T. brucei, no difference occurs between the COII mRNA levels of insect and mammalian stages of T. cruzi.

Amino Acid Sequence↗
PubMed1994

Characterization of a Trypanosoma cruzi poly(A)-binding protein and its genes.

We have characterized the biochemical properties of a 66-kDa poly(A)-binding protein (PABP1) in the protozoan Trypanosoma cruzi and isolated two classes of cDNAs encoding the protein. In concordance, Southern blots showed the presence of 2 gene copies. The two cDNA classes differ in the length of adenosine-rich segments in the 5' untranslated region and in point changes scattered throughout the sequence, but their 1650-bp open reading frames encode identical proteins. A single mRNA of 5.5 kb was detected, indicating that the noncoding regions are unusually long. Both the mRNA and the protein are constitutively expressed in all stages of T. cruzi life cycle. The biochemical properties and sequence comparisons show that the T. cruzi PABP1 is similar to the PABP1 of other eukaryotic organisms. These results indicate that PABP1 has been conserved throughout eukaryotic evolution.

Amino Acid Sequence↗
PubMed1994

Characterization of kinetoplast DNA minicircles in Trypanosoma rangeli.

Kinetoplast DNA, the mitochondrial DNA of kinetoplastid protozoans, is a network of interlocked minicircles and maxicircles. We analyzed the sequence organization of minicircle DNAs in the El Tocuyo strain and the San Augustin clone B6 of Trypanosoma rangeli. The frequencies of different minicircle types, as defined by the number of 136-bp conserved regions (CRs), are different in the two strains. About half of the 1.7-kb T. rangeli El Tocuyo minicircles have 1 CR and most of the others have 2. In contrast, most of the 1.6-kb T. rangeli San Augustin minicircles have 2 CRs, while some have four. The CR contains a replication origin at one end and is conserved both within and between the two strains. Comparisons of the T. rangeli El Tocuyo and T. rangeli San Augustin minicircle CRs with minicircle CRs of other kinetoplastid species reveal that they are most similar to those of Trypanosoma cruzi.

Animals↗
PubMed1994

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.

Amino Acid Sequence↗
PubMed1994

A strand bias occurs in point mutations associated with variant surface glycoprotein gene conversion in Trypanosoma rhodesiense.

We previously described a bloodstream Trypansoma rhodesiense clone, MVAT5-Rx2, whose isolation was based on its cross-reactivity with a monoclonal antibody (MAb) directed against a metacyclic variant surface glycoprotein (VSG). When the duplicated, expressed VSG gene in MVAT5-Rx2 was compared with its donor (basic copy) gene, 11 nucleotide differences were found in the respective 1.5-kb coding regions (Y. Lu, T. Hall, L. S. Gay, and J. E. Donelson, Cell 72:397-406, 1993). Here we describe a characterization of two additional bloodstream trypanosome clones, MVAT5-Rx1 and MVAT5-Rx3, whose VSGs are expressed from duplicated copies of the same donor VSG gene. The three trypanosome clones each react with the MVAT5-specific MAb, but they have different cross-reactivities with a panel of other MAbs, suggesting that their surface epitopes are similar but nonidentical. Each of the three gene duplication events occurs at a different 5' crossover site within a 76-bp repeat and is associated with a different set of point mutations. The 35, 11, and 28 point mutations in the duplicated VSG coding regions of Rx1, Rx2, and Rx3, respectively, exhibit a strand bias. In the sense strand, of the 74 total mutations generated in the three duplications, 54% are A-to-G or G-to-A (A:G) transitions and 7% are C:T transitions, while 26% are C:A transversions and 13% are C:G transversions. No T:G or T:A transversions occurred. Possible models for the generation of these point mutations are discussed.

Amino Acid Sequence↗