Detection and quantitation of human respiratory syncytial virus (RSV) using minigenome cDNA and a Sindbis virus replicon: a prototype assay for negative-strand RNA viruses

African trypanosomes undergo antigenic variation of their variant surface glycoprotein (VSG) coat to avoid being killed by their mammalian hosts. The active VSG gene is located in one of many telomeric expression sites. Replacement of the VSG gene in the active site or switching between expression sites can give rise to a new VSG coat. To study Trypanosoma brucei VSG expression site inactivation rather than VSG gene switching, it is useful to have an in vitro negative-selection system independent of the VSG. We have achieved this aim by using a viral thymidine kinase (TK) gene. Following integration of the TK gene downstream of the 221a VSG expression site promoter, transformant cell lines became sensitive to the nucleoside analog 1-(2-deoxy-2-fluoro-8-D-arabinofuranosyl)-5-iodouracil. These TK trypanosomes were able to revert to resistance at a rate approaching 10(-5) per cell per generation. The majority of revertants expressed a new VSG gene even though there had been no selection against the VSG itself. Analysis of these switched variants showed that some had shut down TK expression via an in situ expression site switch. However, most variants had the complete 221 expression site deleted and another VSG expression site activated. We speculate that a new VSG expression site cannot switch on without inactivation of the old site.     

Localization of the modified base J in telomeric VSG gene expression sites of Trypanosoma brucei

African trypanosomes such as Trypanosoma brucei undergo antigenic variation in the bloodstream of their mammalian hosts by regularly changing the variant surface glycoprotein (VSG) gene expressed. The transcribed VSG gene is invariably located in a telomeric expression site. There are multiple expression sites and one way to change the VSG gene expressed is by activating a new site and inactivating the previously active one. The mechanisms that control expression site switching are unknown, but have been suggested to involve epigenetic regulation. We have found previously that VSG genes in silent (but not active) expression sites contain modified restriction endonuclease cleavage sites, and we have presented circumstantial evidence indicating that this is attributable to the presence of a novel modified base beta-D-glucosyl-hydroxymethyluracil, or J. To directly test this, we have generated antisera that specifically recognize J-containing DNA and have used these to determine the precise location of this modified thymine in the telomeric VSG expression sites. By anti J-DNA immunoprecipitations, we found that J is present in telomeric VSG genes in silenced expression sites and not in actively transcribed telomeric VSG genes. J was absent from inactive chromosome-internal VSG genes. DNA modification was also found at the boundaries of expression sites. In the long 50-bp repeat arrays upstream of the promoter and in the telomeric repeat arrays downstream of the VSG gene, J was found both in silent and active expression sites. This suggests that silencing results in a gradient of modification spreading from repetitive DNA flanks into the neighboring expression site sequences. In this paper, we discuss the possible role of J in silencing of expression sites.     

Stable expression of mosaic coats of variant surface glycoproteins in Trypanosoma brucei

The paradigm of antigenic variation in parasites is the variant surface glycoprotein (VSG) of African trypanosomes. Only one VSG is expressed at any time, except for short periods during switching. The reasons for this pattern of expression and the consequences of expressing more than one VSG are unknown. Trypanosoma brucei was genetically manipulated to generate cell lines that expressed two VSGs simultaneously. These VSGs were produced in equal amounts and were homogeneously distributed on the trypanosome surface. The double-expressor cells had similar population doubling times and were as infective as wild-type cells. Thus, the simultaneous expression of two VSGs is not intrinsically harmful.     

A Trypanosoma brucei bloodstream form mutant deficient in ornithine decarboxylase can protect against wild-type infection in mice

A Trypanosoma brucei bloodstream mutant in which both copies of the ornithine decarboxylase (ODC) gene were knocked out (ODC mutant) was used to determine the biological functions of ODC in T. brucei. Growth of the mutant cells ceased within 12-24 h in regular culture medium deficient in polyamines, but could be rescued by supplementation with 1 mM putrescine. A mouse model of T. brucei infection was used to determine whether the mutant was still infective and was found to develop either extremely low or undetectable levels of parasitemia, suggesting that in T. brucei, ODC activity is essential for establishing an infection. Furthermore, when these mice were subsequently challenged with wild-type T. brucei cells expressing the same variant surface glycoprotein (VSG), they did not develop any parasitemia, indicating that inoculating the mice with the attenuated ODC mutant had conferred protection against challenge by wild-type cells. These results were reproduced in C57BL/6J mice deficient in alpha-beta and gamma-delta T-cell receptors. However, no protection was observed in rag-2 knockout mice deficient in both B and T lymphocytes or in C57BL/10J mice deficient only in B lymphocytes. The results thus suggest that the ODC mutant could induce a T-lymphocyte-independent but B-lymphocyte-dependent immunity against wild-type cells of the same VSG. Such a mechanism of immunity has been elicited only by live T. brucei cells, but not by isolated VSGs or radiation-killed trypanosomes. This ODC mutant may thus represent a genuinely attenuated T. brucei bloodstream form capable of immunizing mammals against infections by African trypanosomes of the same VSG subtype without causing detectable infection by itself. The observation also raises the interesting likelihood that the in vivo treatment of T. brucei bloodstream forms with alpha-DL-difluoromethylornithine is a de facto attenuation of the parasitic organisms, which may very well result in B-lymphocyte-dependent host immune responses to subsequent infections by parasites of the same VSG subtypes.     

Partitioning of large and minichromosomes in Trypanosoma brucei

The Trypanosoma brucei nuclear genome contains about 100 minichromosomes of between 50 to 150 kilobases and about 20 chromosomes of 0.2 to 6 megabase pairs. Minichromosomes contain nontranscribed copies of variant surface glycoprotein (VSG) genes and are thought to expand the VSG gene pool. Varying VSG expression allows the parasite to avoid elimination by the host immune system. The mechanism of inheritance of T. brucei chromosomes was investigated by in situ hybridization in combination with immunofluorescence. The minichromosome population segregated with precision, by association with the central intranuclear mitotic spindle. However, their positional dynamics differed from that of the large chromosomes, which were partitioned by kinetochore microtubules.     

Both IgM and IgG anti-VSG antibodies initiate a cycle of aggregation-disaggregation of bloodstream forms of Trypanosoma brucei without damage to the parasite

Bloodstream forms of Trypanosoma brucei, when aggregated in the presence of either acute immune plasma, acute immune serum, purified IgM anti-VSG antibodies or purified IgG anti-VSG antibodies, subsequently disaggregated with a t1/2 for disaggregation of 15 min at 37 degrees C as long as the trypanosomes were metabolically active at the beginning of the experiment and maintained during the experiment in a suitable supporting medium. The t1/2 for disaggregation was found to be directly dependent upon temperature and inversely proportional to the antibody concentration. The trypanosomes were always motile and metabolically active during aggregation and after disaggregation and were fully infective for a mammalian host following disaggregation as well as able to grow and divide normally during axenic culture. The disaggregation was strictly energy dependent and was inhibited when intracellular ATP levels were reduced by salicylhydroxamic acid or following addition of oligomycin while respiring glucose. In addition the process of disaggregation was dependent upon normal endosomal activity as evidenced by its sensitivity to a wide variety of inhibitors of various endosomal functions. Disaggregation was not due to separation of immunoglobulin chains by either disulphide reduction or disulphide exchange reactions and gross proteolytic cleavage of the immunoglobulins attached to the surface of the parasite was not detected. In addition, gross cleavage or release of the VSG from the surface of the cell did not occur during disaggregation but proteolytic cleavage of a small proportion of either the VSG or the immunoglobulins could not be eliminated from consideration. Finally the mechanism of disaggregation was found to be a regulated process, independent of Ca2+ movements but dependent upon the activity of protein kinase C or related kinases and inhibited by the activity of protein kinase A as evidenced by the effects of a panel of inhibitors and cAMP analogues on the process of disaggregation. The mechanism of disaggregation displayed by trypanosomes aggregated by anti-VSG antibody is proposed to form part of the parasite's defence against the host immune system and functions to aid survival of trypanosomes in the presence of antibody in the host prior to the occurrence of a VSG switching event.     

Construction and characterization of a radio-iodinatable mutant of recombinant human CD4

Drug-selected intrachromosomal gene amplification by breakage-fusion-bridge (BFB) cycles is well documented in mammalian cells, but factors governing this mechanism are not clear. Here, we show that only some clastogenic drugs induce drug resistance through intrachromosomal amplification. We strictly correlate triggering of BFB cycles to induction of fragile site expression. We demonstrate a dual role for fragile sites in intrachromosomal amplification: a site telomeric to the selected gene is involved in initiation, while a centromeric site defines the size and organization of early amplified units. The positions of fragile sites relative to boundaries of amplicons found in human cancers support the hypothesis that fragile sites play a key role in the amplification of at least some oncogenes during tumor progression.     

The ubiquitin-conjugating enzyme Rad6 (Ubc2) is required for silencing in Saccharomyces cerevisiae

It has been previously shown that genes transcribed by RNA polymerase II (RNAP II) are subject to position effect variegation when located near yeast telomeres. This telomere position effect requires a number of gene products that are also required for silencing at the HML and HMR loci. Here, we show that a null mutation of the DNA repair gene RAD6 reduces silencing of the HM loci and lowers the mating efficiency of MATa strains. Likewise, rad6-delta reduces silencing of the telomere-located RNAP II-transcribed genes URA3 and ADE2. We also show that the RNAP III-transcribed tyrosyl tRNA gene, SUP4-o, is subject to position effect variegation when located near a telomere and that this silencing requires the RAD6 and SIR genes. Neither of the two known Rad6 binding factors, Rad18 and Ubr1, is required for telomeric silencing. Since Ubrl is the recognition component of the N-end rule-dependent protein degradation pathway, this suggests that N-end rule-dependent protein degradation is not involved in telomeric silencing. Telomeric silencing requires the amino terminus of Rad6. Two rad6 point mutations, rad6(C88A) and rad6(C88S), which are defective in ubiquitin-conjugating activity fail to complement the silencing defect, indicating that the ubiquitin-conjugating activity of RAD6 is essential for full telomeric silencing.     

(+)-7-Deaza-5''-noraristeromycin as an anti-trypanosomal agent

The (+)-enantiomer of 7-deaza-5'-noraristeromycin (4) has been found to show IC50 values ranging from 0.16 to 5.3 microM against four strains of African trypanosomes, one Trypanosoma brucei brucei isolate, and several clinical isolates of Trypanosoma brucei rhodesiense (agent of east African sleeping sickness), including a multidrug resistant clone of one isolate. While this compound was originally designed to inhibit S-adenosyl-L-homocysteine hydrolase, it has been found to have no effect on this enzyme.