Richard O. Williams

Relationship between multiple copies of a T. brucei variable surface glycoprotein gene whose expression is not controlled by duplication

Unlike many other T. brucei variable surface glycoprotein (VSG) genes, the IITat 1.3 gene is not duplicated when it is expressed. Analysis of the multiple copies of this gene present in all IITaR 1 trypanosome clones by restriction enzyme mapping and sequencing shows that the expressed copy may have arisen by duplication and transposition to a telomeric site, as is observed for those VSG genes whose expression is linked to duplication. The existence of a mechanism selecting between a number of complete telomeric VSG gene copies for expression is implied by these results. Comparisons of the nontelomeric copies of the IITat 1.3 gene are consistent with involvement of gene duplication and mutational drift in the evolution of new VSG genes.

Kinetoplast DNA minicircles of Trypanosoma brucei share regions of sequence homology.

Abstract Kinetoplast DNA (kDNA) of Trypanosoma brucei consists of massive networks of 10,000 or more interlocked molecules of maxicircle DNA (about 23 kb each) and minicircle DNA (1.1 kb each). Individual minicircle DNA molecules were released from the network by digestion with HaeIII, HpaII, AluI, HhaI, PstI, or HindIII and cloned in E. coli via the plasmid pBR322 and the poly(dG):poly(dC) tailing technique or the DNA ligase technique. The cloned minicircle DNA molecules were compared (i) by two types of filter hybridization, (ii) by renaturation kinetics, and (iii) by heteroduplex analysis. The sequence complexity of total network kDNA is about 300 times that of a single cloned minicircle kDNA molecule. The filter hybridizations and heteroduplex analyses suggest that minicircle molecules possess sequences in common with each other. The renaturation kinetics indicates that these homologous regions comprise about one-fourth of the 1.1-kb minicircle molecule. Therefore each minicircle molecule appears to have about one-fourth of its sequence in common with a large percentage of the total minicircle population and the remaining three-fourths in common with about 1 out of 300 minicircle molecules.

Detection and characterization of DNA polymerase from Trypanosoma brucei

The predominant DNA polymerase activity has been isolated from the parasitic flagellated protozoan, Trypanosoma brucei. Like mammalian DNA polymerase-alpha the trypanosome DNA polymerase is of large molecular weight (S, 6--8), is resistant to thermal denaturation, is sensitive to N-ethylmaleimide, and is inhibited by high ionic strength. However, specific antisera that cross-react with mammalian DNA polymerase-alpha from different species fail to cross-react with the trypanosome polymerase.

Cloning and analysis of Trypanosoma (Nannomonas) congolense ILNat 2.1 VSG gene

Genes encoding various Trypanosoma (Trypanozoon) brucei variable surface glycoproteins (VSGs) show considerable conservation among different members of this species, known as isotypes. The occurrence of isotypes in other salivarian trypanosomes has not been well documented. We have cloned sequences encoding Trypanosoma (Nannomonas) congolense ILNat 2.1 VSG, and used it in DNA blot hybridization analyses of this and other T. congolense clones originating from geographically separate regions of East Africa. The data indicate that the expression of ILNat 2.1 VSG gene proceeds by duplicative transposition resulting in the presence of an extra expression-linked copy in the expressing clones examined. Furthermore the ILNat 2.1 VSG gene sequence is absent or has greatly diverged, in all other T. (N.) congolense clones that belong to different serodemes. This suggests that some T. (N.) congolense VSGs may be limited to their respective antigen repertoires. The data are discussed in the light of their implications for antigenic variation in T. (N.) congolense, and parasite epidemiology.

Shared surface epitopes among trypanosomes of the same serodeme expressing different variable surface glycoprotein genes.

African trypanosomes evade the immune response of the mammalian host by undergoing antigenic variation, caused by sequence changes in a variable surface glycoprotein (VSG). The majority of trypanosome clones analyzed thus far are not known to share surface exposed epitopes or express appreciably homologous VSGs. We show here that four clones of Trypanosoma brucei from the same serodeme express different VSGs and share exposed epitopes to varying degrees, as defined by monoclonal antibodies. Rabbit antiserum against any one of the four VSGs recognizes epitopes present on all four trypanosomes in live cell immunofluorescence assay. The expressed VSGs are partially homologous at the N-terminus with multiple point substitutions of amino acids which distinguish each of the four VSGs. The genes coding for these VSGs are members of one gene family and an expression-linked copy with a unique restriction map is present in each trypanosome. Analysis of the ontogeny of the expressed genes should reveal mechanisms of evolution in trypanosome variable antigen repertoires.

Microbial Surface Elements: The Case of Variant Surface Glycoprotein (VSG) Genes of African Trypanosomes

Trypanosoma brucei and its related species (T. congolense, T. equiperdum and T. vivax) are hemoparasitic, flagellated protozoa that are the causative pathogenic agents of African sleeping sickness in humans (T. brucei rhodesiense and T. brucei gambiense) and in domestic animals. T. brucei is transmitted by the tsetse fly (genus Glossina) thereby largely restricting these diseases to the African continent. During its life cycle in the fly and subsequently in the mammalian host, T. brucei normally appears to be pleomorphic in nature exhibiting several distinctive morphological forms (1). Slender bloodstream forms are present in the initial stages of an infection while nondividing, stumpy forms accumulate at later times. The stumpy forms are ingested by the fly and are transformed into procyclic forms (the only stage lacking a defined surface coat) in the midgut of the fly (2,3). The procyclic forms eventually migrate into the salivary glands where they differentiate into a heterogeneous population of metacyclic forms which are transmitted back to the host to complete the life cycle. Evolution has imparted intricate mechanisms to the trypanosomes for maintaining chronic infections in their mammalian hosts, thereby allowing for cyclical transmission via the tsetse fly. This unique phenomenon appears to be manifested by the ability of infectious trypanosomes to temporally alter this surface antigen structure thereby evading the host’s immune response (1,4-7). The net result is that T. brucei is a brilliant parasite.