Michael Milhausen

Tubulin genes are tandemly linked and clustered in the genome of trypanosoma brucei

We have isolated cDNA and genomic clones containing alpha- and beta-tubulin genes from Trypanosoma brucei. Each clone has been mapped, and the identity of the tubulin genes has been established by cross-hybridization with cloned chicken tubulin genes and by hybridization-selection and translation of trypanosome tubulin mRNA. In contrast with the dispersed organization of tubulin genes in other organisms, trypanosome alpha- and beta-tubulin genes are physically linked and clustered in tandem repeats of approximately 13-17 copies per haploid genome of alternating alpha- and beta-tubulin sequences.

Identification of a small rna containing the trypanosome spliced leader: A donor of shared 5′ sequences of trypanosomatid mRNAs?

Abstract The 35 nucleotide spliced leader (SL) sequence is found on the 5′ end of numerous trypanosome mRNAs, yet the tandemly organized reiteration units encoding this leader are not detectably linked to any of these structural genes. Here we report the presence of a class of discrete small SL RNA molecules that are derived from the genomic SL reiteration units of Trypanosoma brucei, Trypanosoma cruzi, and Leptomonas collosoma. These small SL RNAs are 135, 105, and 95 nucleotides, respectively, and contain a 5′-terminal SL or SL-like sequence. S1 nuclease analyses demonstrate that these small SL RNAs are transcribed from continuous sequence within the respective SL reiteration units. With the exception of the SL sequence and a concensus donor splice site immediately following it, these small RNAs are not well conserved. We suggest that the small SL RNAs may function as a donor of the SL sequence in an intermolecular process that places the SL at the 5′ terminus of many trypanosomatid mRNAs.

A developmentally regulated cysteine protease gene family in Haemonchus contortus

The nucleotide sequence of a gene encoding a 35-kDa thiol protease of the parasitic nematode Haemonchus contortus has been determined. The gene, designated AC-2, shares 97% nucleotide sequence identity and 98% amino acid identity with previously characterized AC-1 cDNAs encoding the thiol protease. The AC-2 gene spans 8 kb and appears to contain 11 introns, ranging in size from 57 bp to over 5.2 kb. One of the introns interrupts the proposed active site region that is conserved between the H. contortus protease and the related thiol proteases cathepsin B and papain. Southern blot hybridization experiments indicate that the protease is encoded by a small gene family in H. contortus. Rabbit antisera prepared against the recombinant protein react on Western blots with 35 and 37-kDa proteins of adult worms. These proteins were not detectable by Western blot analysis in three larval parasitic developmental stages of H. contortus. Northern blot hybridizations indicate that mRNA transcripts for the gene family are present at low levels in a mixed population of third- and fourth-stage larvae but highly abundant in adult worms. Expression of the protease correlates with blood-feeding and suggests a role for the protease in blood digestion.

The Istar 1 Serodeme of Trypanosoma brucei: Development of a New Serodeme

An extensive serodeme of sequentially-isolated antigenic variants of African trypanosomes has been produced from both syringe-passaged and cyclically-transmitted Trypanosoma brucei of the IsTaR 1 clone derived from EATRO 164. The majority of the antigenic variants were isolated from chronically-infected deer mice (Peromyscus leucopus). The pattern of parasitemias during the course of infections initiated with syringe-passaged trypanosomes differed from those initiated with cyclically-transmitted trypanosomes. Trypanosome populations from syringe-passaged (192) and cyclically-transmitted (31) clones were each amplified by growth in lethally-irradiated mice and cryopreserved for retrospective analysis. Five clones derived from a single deer mouse during the first 44 days of infection, and 2 clones derived from an acutely-infected rat were established from these amplified populations. Homogeneous populations were grown in lethally-irradiated rats and mice for antigenic analysis purification of variant-specific glycoprotein. Six of the 7 clones were distinct variants by immunological criteria using antisera derived from whole cells or purified surface glycoproteins. Two clones, one derived from the acutely-infected rat, and the other from the first parasitemia in a chronic infection that was initiated with the former clone, were immunologically identical. Production of these clones established a well-defined serodeme that will allow detailed analysis of antigenic variation.

Molecular characterization of initial variants from the IsTat I serodeme of Trypanosoma brucei

Variant surface glycoproteins (VSGs) were isolated from variant antigen types (VATs) of the IsTat 1 serodeme. Molecular weight and isoelectric focusing analysis demonstrate that seven early VSGs possess properties generally attributed to VSGs isolated from other trypanosome serodemes. Six of the seven VSGs characterized are distinct from one another, while two (D and 1) appear identical. The presence of VSG specific mRNA in corresponding VATs was demonstrated by in vitro translation of RNA from each of the VATs, followed by immunoprecipitation with homologous and heterologous antisera. Hybridization of VSG cDNA clones with RNA from each VAT confirm that VSG mRNA is present only in homologous VATs and verifies the transcriptional control of these VSG genes. The two VATs D and 1 express indistinguishable VSGs by a variety of biochemical criteria, as well as by reactivity with 24 monoclonal antibodies. The VSG mRNAs in VATs D and 1 also appear identical. However, this identity is not reflected at the genomic level. Data is presented which establishes that DNA rearrangements can occur around both expressed and non-expressed VSG genes without qualitatively affecting VSG gene expression.

Genomic organization of Trypanosoma brucei variant antigen gene families in sequential parasitemias.

cDNA libraries were made from mRNA purified from each of seven sequentially isolated variant antigen types (VATs) of the IsTat 1 serodeme. Plasmids containing variant surface glycoprotein (VSG) sequences corresponding to each of the isolates were used in Southern analyses to examine the genomic organization of VSG nucleotide sequences. In most cases, cells expressing a given VSG were shown to have an extra copy of the corresponding VSG gene. In one case an expression-linked copy (ELC) was not detectable. VSG gene rearrangements not obviously correlated with the expression of homologous sequences were detected in four of six VSG gene families. Thus, even cDNAs which detected an ELC revealed additional genomic reorganization in regions flanking VSG sequences. The cells used to initiate the chronic infection expressed the same VSG as those isolated from the first parasitemia. The extent of genomic rearrangement observed between these two sequentially derived populations was comparable to that observed between any of the other serially derived VATs. Thus, within a short period of time and in the absence of detectable antigenic variation, the amount of genetic flux in sequences associated with VSG genes can be substantial.

Localization of Toxoplasma gondii in feline intestinal tissue using PCR.

Gametogony of Toxoplasma gondii occurs only in the epithelial cell layers within the intestine of the definitive feline host. Infected feline intestine is required in order to study the physiology, histology, and molecular biology of the gametogenic stages. Therefore, we set out to devise a rapid, conservative, and reproducible technique to determine which portions of the intestine were infected. Several methods of collecting and processing infected material were assessed for their ability to detect T. gondii. Infected and uninfected intestines from domestic cats were used to produce nitrocellulose lift impressions along the entire small intestine. The nitrocellulose was analyzed for the presence of T. gondii DNA using polymerase chain reaction (PCR) primers specific for the T. gondii alpha-tubulin gene. In addition, mucosal tissue scrapings, derived from segments of intestinal tissue, were used to isolate DNA and RNA for subsequent PCR and reverse transcriptase PCR analysis, respectively. The nitrocellulose impression lift method demonstrated distribution of parasite throughout the intestine. Histological staining and indirect immunofluorescence antibody analysis of sections obtained from the same infected tissue confirmed the presence of T. gondii intraepithelial stages. Comparison among the different techniques indicates that the nitrocellulose impression lift technique proved to be effective for easily and quickly assessing presence of T. gondii in infected tissue. This technique does not require a significant amount of the experimental material.

ANALYSIS OF THE HUMORAL RESPONSES OF TOXOPLASMA GONDII–INFECTED CATS USING IMMUNOFLUORESCENT ASSAYS WITH TACHYZOITE, BRADYZOITE, AND GAMETOGENIC STAGES

We investigated levels of Toxoplasma gondii–specific antibodies present in sera, intestinal secretions, and fecal extracts obtained from cats following primary and challenge infections. Antibodies specific to T. gondii tachyzoites, bradyzoites, sporozoites, and enteroepithelial stages were detected by indirect immunofluorescence assay. Enteroepithelial stage–specific antibodies were detected in serum as early as 2 wk after infection, whereas antibodies from intestinal secretions did not appear until 3 wk following infection. The T. gondii–specific IgG and IgA antibodies were present in serum, but only specific IgA antibodies were detected in the intestinal secretions. Serum IgG bound to tachyzoites, bradyzoites, sporozoites, and enteroepithelial stages of T. gondii, whereas serum IgA bound strongly to enteroepithelial stages but only weakly to tachyzoites and bradyzoites. IgA from intestinal secretions bound to antigens on all enteroepithelial stages and the distal tips of sporozoites and bradyzoites but did not bind to tachyzoites. IgA present in fecal extracts also bound to enteroepithelial stages of T. gondii. Toxoplasma gondii infection in cats induces the production of antibodies that bind with all forms of the parasite, including the enteroepithelial stages. Comparison of the staining patterns of T. gondii stages for serum and intestinal secretion IgA indicated differences. Thus, the intestinal antibody immune response may be uniquely focused on the intestinal stages relative to the circulating antibodies, resulting in a compartmentalization of the humoral response.