Antigenic Variation and Allelic Exclusion

AbstractCells often express only one gene from a set of two or more. African trypanosomes appear to accomplish this monoallelic expression by segregating the selected gene into a specific nuclear body. The possibility that such a structure might explain monoallelic expression in other multigene systems is discussed here

The isolation of plasmids containing DNA complementary to messenger RNA for variant surface glycoproteins of Trypanosoma brucei.

We have isolated poly(A)+ RNA from four antigenic variants (117, 118, 121, 221) of one clone of Trypanosoma brucei. Translation of these poly(A)+ RNAs in a rabbit reticulocyte lysate gave rise to proteins that could be precipitated with antisera against homologous variant surface glycoprotein, the protein responsible for antigenic variation in trypanosomes. From the electrophoretic mobility of these in vitro products in sodium dodecyl sulphate (SDS) gels we infer that variant surface glycoproteins (VSGs) are made as pre-proteins, which require trimming to yield mature VSGs. The total translation products from the four poly(A)+ RNAs produced a complex set of bands on SDS gels, which only differed in the region where the variant pre-glycoproteins migrated. The only detectable variation in the messenger RNA populations of these variants is, therefore, in the messenger RNA for variant pre-glycoproteins. We have made duplex DNA copies of these poly(A)+ RNAs, linked the complementary DNA to plasmid pBR322 by GC tailing and cloned this recombinant DNA in Escherichia coli. Colony hybridization with complementary DNA made on poly(A)+ RNA showed that 7--10% of the colonies contained DNA that hybridized only with the homologous probe. Plasmid DNA was isolated from ten such colonies (two or three of each variant complementary DNA), bound to diazobenzyloxymethyl-cellulose (DBM) paper and used to select complementary messenger RNA from total poly(A)+ RNA by hybridization. In eight cases the RNA recovered from the filter gave variant pre-glycoprotein as the predominant product of in vitro translation. Poly(A)+ RNA from each of the variants only hydridized to the homologous complementary DNA in filter hybridizations. Each trypanosome variant, therefore, contains no detectable messenger RNAs for the three heterologous variant-specific glycoproteins tested. We conclude from this lack of cross-hybridization that antigenic diversity in trypanosomes, unlike antibody diversity in mammals, does not involve the linkage of a repertoire of genes for the variable N-terminal half to a single gene for the C-termina

Analysis of cosmid clones of nuclear DNA from Trypanosome brucei shows that the genes for variant surface glycoproteins are clustered in the genome.

Trypanosoma brucei contains more than a hundred genes coding for the different variant surface glycoproteins (VSGs). Activation of some of these genes involves the duplication of the gene (the basic copy or BC) and transposition of the duplicate to an expression site (yielding the expression-linked copy or ELC). We have cloned large fragments of genomic DNA in cosmid vectors in Escherichia coli. Cosmids containing the BCs of genes 117, 118 and 121 were readily obtained, but DNA containing the ELCs was strongly selected against in the cosmid and plasmid cloning systems used. We have analysed the distribution of VSG genes in the genome using probes for the sequences at the edges of the transposed segment which are partially homologous among these genes. In genomic cosmid clone banks, about 9% of all colonies hybridize with probes from the 5'- and 3'-edges of the transposed segment, showing that these sequences are linked in the genome. Moreover, the 117 and 118 BC cosmids contain several additional putative VSG genes in tandem, as deduced from hybridization and sequence analyses. We conclude that the VSG genes are highly clustered and share common sequences at the borders of the transposed segment

Formation of linear inverted repeat amplicons following targeting of an essential gene in Leishmania

Attempts to inactivate an essential gene in the protozoan parasite Leishmania have often led to the generation of extra copies of the wild-type alleles of the gene. In experiments with Leishmania tarentolae set up to disrupt the gene encoding the J-binding protein 1 (JBP1), a protein binding to the unusual base β-d-glucosyl-hydroxymethyluracil (J) of Leishmania, we obtained JBP1 mutants containing linear DNA elements (amplicons) of ∼100 kb. These amplicons consist of a long inverted repeat with telomeric repeats at both ends and contain either the two different targeting cassettes used to inactivate JBP1, or one cassette and one JBP1 gene. Each long repeat within the linear amplicons corresponds to sequences covering the JBP1 locus, starting at the telomeres upstream of JBP1 and ending in a ∼220 bp sequence repeated in an inverted (palindromic) orientation downstream of the JBP1 locus. We propose that these amplicons have arisen by a template switch inside a DNA replication fork involving the inverted DNA repeats and helped by the gene targeting

Reduction of Oxidative Stress in Chronic Kidney Disease Does Not Increase Circulating alpha-Klotho Concentrations

The CKD-associated decline in soluble α-Klotho levels is considered detrimental. Some in vitro and in vivo animal studies have shown that anti-oxidant therapy can upregulate the expression of α-Klotho in the kidney. We examined the effect of anti-oxidant therapy on α-Klotho concentrations in a clinical cohort with mild tot moderate chronic kidney disease (CKD). We performed a post-hoc analysis of a prospective randomized trial involving 62 patients with mild to moderate CKD (the ATIC study), all using an angiotensin-converting enzyme inhibitor (ACEi) or angiotensin receptor blocker (ARB) for 12 months. On top of that, the intervention group received anti-oxidative therapy consisting of the combination of pravastatin (40 mg/d) and vitamin E (α-tocopherol acetate, 300 mg/d) while the placebo was not treated with anti-oxidants. α-Klotho concentrations were measured at baseline and after 12 months of anti-oxidant therapy. Data were analysed using T-tests and Generalized Estimating Equations, adjusting for potential confounders such as vitamin D, parathyroid hormone, fibroblast-growth-factor 23 (FGF23) and eGFR. The cohort existed of 62 patients with an eGFR (MDRD) of 35 ± 14 ml/min/1.72 m2, 34 were male and mean age was 53.0 ± 12.5 years old. Anti-oxidative therapy did successfully reduce oxLDL and LDL concentrations (P <0.001). α-Klotho concentrations did not change in patients receiving either anti-oxidative therapy (476.9 ± 124.3 to 492.7 ± 126.3 pg/mL, P = 0.23) nor in those receiving placebo 483.2 ± 142.5 to 489.6 ± 120.3 pg/mL, P = 0.62). Changes in α-Klotho concentrations were not different between both groups (p = 0.62). No evidence was found that anti-oxidative therapy affected α-Klotho concentrations in patients with mild-moderate CKD

The protein that binds to DNA base J in trypanosomatids has features of a thymidine hydroxylase

© 2007 The Author et al. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. The definitive version was published in Nucleic Acids Research 35 (2007): 2107-2115, doi:10.1093/nar/gkm049.Trypanosomatids contain an unusual DNA base J (ß-D-glucosylhydroxymethyluracil), which replaces a fraction of thymine in telomeric and other DNA repeats. To determine the function of base J, we have searched for enzymes that catalyze J biosynthesis. We present evidence that a protein that binds to J in DNA, the J-binding protein 1 (JBP1), may also catalyze the first step in J biosynthesis, the conversion of thymine in DNA into hydroxymethyluracil. We show that JBP1 belongs to the family of Fe2+ and 2-oxoglutarate-dependent dioxygenases and that replacement of conserved residues putatively involved in Fe2+ and 2-oxoglutarate-binding inactivates the ability of JBP1 to contribute to J synthesis without affecting its ability to bind to J-DNA. We propose that JBP1 is a thymidine hydroxylase responsible for the local amplification of J inserted by JBP2, another putative thymidine hydroxylase.This work was funded by a grant from the Netherlands Organization for Scientific Research and Chemical Sciences (NWO-CW) to P.B., NIH grant A1063523 to R.S. and NIH grant GM063584 to R.P.H