Control of spontaneous activity during development

The African trypanosome Trypanosoma brucei expresses the active variant surface glycoprotein (VSG) gene in a telomeric VSG gene expression site. We have generated trypanosomes with a neomycin resistance gene inserted behind an active VSG gene expression site promoter, and a hygromycin resistance gene behind a silent one. By alternating drug selection, we could select for trypanosomes that had switched between the two marked VSG gene expression sites. Surprisingly, trypanosomes that had activated a new VSG gene expression site had often lost the old one. Using polymerase chain reaction (PCR), we screened large numbers of switched trypanosomes and found that sequences lost invariably included the drug marker near the promoter, as well as the telomeric VSG gene many tens of kilobases away. We postulate that stable activation of a new expression site requires silencing of the old one. If silencing does not occur at a sufficient rate by normal switch-off, stable activation of the new site can only occur if the old site is lost in random deletion events. The fact that we pick up these normally infrequent deletions, indicates that inactivation of the old VSG expression site could be rate limiting during switching in our strain of T. brucei.     

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.     

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.     

Imaging herpes virus thymidine kinase gene transfer and expression by positron emission tomography

We report a series of studies that assess the feasibility and sensitivity of imaging of herpes virus type one thymidine kinase (HSV1-tk) gene transfer and expression with [124I]-5-iodo-2'-fluoro-1-beta-D-arabinofuranosyluracil ([124I]-FIAU) and positron emission tomography (PET) and the ability of [124I]-FIAU-PET imaging to discriminate different levels of HSV1-tk gene expression. Studies were performed in rats bearing multiple s.c. tumors derived from W256 rat carcinoma and RG2 rat glioma cells. In the first set, we tested the sensitivity of [124I]-FIAU-PET imaging to detect low levels of HSV1-tk gene expression after retroviral-mediated gene transfer. HSV1-tk gene transduction of one of preestablished wild-type W256 tumor in each animal was accomplished by direct intratumoral injection of retroviral vector-producer cells (W256-->W256TK* tumors). Tumors produced from W256 and W256TK+ cells served as the negative and positive control in each animal. Highly specific images of [124I]-FIAU-derived radioactivity were obtained in W256TK* tumors (that were transduced in vivo) and in W256TK+ tumors but not in nontransduced wild-type W256 tumors. The level of "specific" incorporated radioactivity in transduced portions of both W256TK* and W256TK+ tumors was relatively constant between 4 and 50 h. In the second set, we tested whether [124I]-FIAU and PET imaging can measure and discriminate between different levels of HSV1-tk gene expression. Multiple s.c. tumors were produced from wild-type RG2 cells and stably transduced RG2TK cell lines that express different levels of HSV1-tk. A highly significant relationship between the level of [124I]-FIAU accumulation [% injected dose/g and incorporation constant (Ki)] and an independent measure of HSV1-tk expression (sensitivity of the transduced tumor cells to ganciclovir, IC50) was demonstrated, and the slope of this relationship was defined as a sensitivity index. We have demonstrated for the first time that highly specific noninvasive images of HSV1-tk expression in experimental animal tumors can be obtained using radiolabeled 2'-fluoro-nucleoside [124I]-FIAU and a clinical PET system. The ability to image the location (distribution) of gene expression and the level of expression over time provides new and useful information for monitoring clinical gene therapy protocols in the future.     

Lithiasis of the distal ureter: ESWL or URS

Human herpesvirus 8 (HHV-8) is a newly discovered virus closely associated with Kaposi's sarcoma and primary effusion lymphomas. When they occur in patients with AIDS, these B-cell lymphomas frequently harbor another human herpesvirus, Epstein-Barr virus (EBV). To determine the molecular mechanisms of the regulation of early gene expression by the immediate-early gene products of HHV-8 and to assess possible molecular interactions between HHV-8 and EBV, we studied the regulation of the HHV-8 thymidine kinase (TK) promoter in cell lines harboring either or both viruses. The constitutive chloramphenicol acetyltransferase (CAT) activity of the TK promoter was low in all six cell lines tested. A putative immediate-early gene product of HHV-8 ORF50, which is a homolog of EBV BRLF1, was cloned into an expression vector and tested for its transactivating capacity. In the presence of 12-O-tetradecanoyl-phorbol-13-acetate (TPA), the CAT activity of the TK promoter was increased 7- to 720-fold by cotransfection with the ORF50 clone in EBV-producing cell lines (Ramos/AW, P3HR-1, and BC-1) but not in EBV-negative cell lines (BCBL-1 and Ramos), nor in the latently EBV-infected cell line Raji. The TK promoter contains three consensus SP1- and two AP1-binding sites. In electrophoretic mobility shift assays, the cellular factor SP1, but not AP1, was found to bind specifically to the TK promoter. To determine whether the increased CAT activity resulted from the interaction of SP1 with the ORF50 gene product, we introduced mutations into two SP1-binding sites. Both mutated SP1 sites had reduced SP1-binding activity and greatly decreased TK promoter responsiveness to ORF50 transactivation, suggesting that upregulation of TK promoter by ORF50 is SP1 dependent.     

Exploring the active site of herpes simplex virus type-1 thymidine kinase by X-ray crystallography of complexes with aciclovir and other ligands

Antiherpes therapies are principally targeted at viral thymidine kinases and utilize nucleoside analogs, the triphosphates of which are inhibitors of viral DNA polymerase or result in toxic effects when incorporated into DNA. The most frequently used drug, aciclovir (Zovirax), is a relatively poor substrate for thymidine kinase and high-resolution structural information on drugs and other molecules binding to the target is therefore important for the design of novel and more potent chemotherapy, both in antiherpes treatment and in gene therapy systems where thymidine kinase is expressed. Here, we report for the first time the binary complexes of HSV-1 thymidine kinase (TK) with the drug molecules aciclovir and penciclovir, determined by X-ray crystallography at 2.37 A resolution. Moreover, from new data at 2.14 A resolution, the refined structure of the complex of TK with its substrate deoxythymidine (R = 0.209 for 96% of all data) now reveals much detail concerning substrate and solvent interactions with the enzyme. Structures of the complexes of TK with four halogen-containing substrate analogs have also been solved, to resolutions better than 2.4 A. The various TK inhibitors broadly fall into three groups which together probe the space of the enzyme active site in a manner that no one molecule does alone, so giving a composite picture of active site interactions that can be exploited in the design of novel compounds.     

Site-directed mutagenesis clarifies the substrate position within the three-dimensional model of the active site of herpes simplex virus type-1 thymidine kinase

Site-directed mutagenesis was used to experimentally verify the 3D model of the active site of herpes simplex virus type-1 thymidine kinase (HSV 1 TK) obtained by homology modelling. For this purpose, D215 and K317 were replaced by R and G, respectively, at homologous positions in the aciclovir-insensitive bovine herpes virus type-1 thymidine kinase (BHV 1 TK). Wild-type and mutated enzymes were expressed in Escherichia coli using a gene fusion vector and purified to homogeneity. While both mutants had the same Km value for thymidine as the recombinant wild-type enzyme (0.2 microM), Vmax was decreased to 20-25% of the original wild-type value. The recombinant wild-type enzyme was inhibited by the substrate analogue aciclovir with a Ki of 146 microM. Both mutants were able to phosphorylate aciclovir to about the same extent as the wild-type enzyme. These findings suggest that neither D215 nor K317 are directly involved in substrate binding. Therefore, a rearrangement of the 3D model is suggested, concerning the assignment of the substrate-binding site and co-substrate-binding site at the right and left side of the phosphate-binding loop, respectively.     

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.     

A fast method for obtaining highly pure recombinant herpes simplex virus type 1 thymidine kinase

Recombinant Herpes Simplex Virus Type 1 thymidine kinase (TK) was isolated in a fast and gentle two-step procedure from Escherichia coli as a thrombin cleavable fusion protein. The TK was expressed as an inducible glutathione S-acetyl transferase fusion protein and purified in a first step by glutathione affinity chromatography. Proteolytic cleavage of the column bound TK with thrombin led to a truncated enzyme, resulting from two new and hitherto unknown cleavage sites, determined by N-terminal sequencing. In a second step, the TK was further purified from the cleavage products by ATP affinity chromatography, yielding homogeneously pure TK as shown by SDS-PAGE and mass spectrometry. Both the fusion protein and the purified enzyme show enzymatic activity with the same Km value of 0.2 microM for the natural substrate thymidine. Determination of the native molecular weight indicated that the pure enzyme and the fusion protein are biologically active as homodimers. Therefore the recombinant enzyme has the same biochemical characteristics as the viral TK, expressed in infected cells.