Henri G.A.M. van Luenen

G Proteins Are Required for Spatial Orientation of Early Cell Cleavages in C. elegans Embryos

Heterotrimeric G proteins are signal-transducing molecules activated by seven transmembrane domain receptors. In C. elegans, gpb-1 encodes the sole Gbeta subunit; therefore, its inactivation should affect all heterotrimeric G protein signaling. When maternal but no zygotic gpb-1 protein (GPB-1) is present, development proceeds until the first larval stage, but these larvae show little muscle activity and die soon after hatching. When, however, the maternal contribution of GPB-1 is also reduced, spindle orientations in early cell divisions are randomized. Cell positions in these embryos are consequently abnormal, and the embryos die with the normal number of cells and well-differentiated but abnormally distributed tissues. These results indicate that maternal G proteins are important for orientation of early cell division axes, possibly by coupling a membrane signal to centrosome position.

Transposition of the nematode Caenorhabditis elegans Tc3 element in the zebrafish Danio rerio

BACKGROUND Transposable elements of the Tc1/mariner family are found in many species of the animal kingdom. It has been suggested that the widespread distribution of this transposon family resulted from horizontal transmission among different species. RESULTS To test the ability of Tc1/mariner to cross species barriers, as well as to develop molecular genetic tools for studying zebrafish development, we determined the ability of the Tc3 transposon, a member of the Tc1/mariner family, to function in zebrafish. Tc3 transposons carrying sequences encoding the green fluorescent protein (GFP) were able to integrate in the fish genome by transposition. Integrated transposons expressed the GFP marker after germline transmission, and were capable of being mobilized upon introduction of transposase protein in trans. CONCLUSIONS Our findings support models of horizontal transmission of Tc1/mariner elements between species. The work also establishes the basis for a novel method of transposon-mediated genetic transformation and for transposon-mediated genetic screens in zebrafish and other organisms.

Glucosylated Hydroxymethyluracil, DNA Base J, Prevents Transcriptional Readthrough in Leishmania

Some Ts in nuclear DNA of trypanosomes and Leishmania are hydroxylated and glucosylated to yield base J (β-D-glucosyl-hydroxymethyluracil). In Leishmania, about 99% of J is located in telomeric repeats. We show here that most of the remaining J is located at chromosome-internal RNA polymerase II termination sites. This internal J and telomeric J can be reduced by a knockout of J-binding protein 2 (JBP2), an enzyme involved in the first step of J biosynthesis. J levels are further reduced by growing Leishmania JBP2 knockout cells in BrdU-containing medium, resulting in cell death. The loss of internal J in JBP2 knockout cells is accompanied by massive readthrough at RNA polymerase II termination sites. The readthrough varies between transcription units but may extend over 100 kb. We conclude that J is required for proper transcription termination and infer that the absence of internal J kills Leishmania by massive readthrough of transcriptional stops.

Rte-1, a retrotransposon-like element in Caenorhabditis elegans

We have characterized a retrotransposon‐like element (Rte‐1) in C. elegans. It was identified while we were sequencing the pim related kinase‐1 (prk‐1) gene. The element is 3,298 bp long and flanked by a 200 bp direct repeat. 95 bp of the direct repeat are present in the coding region of prk‐1. Rte‐1 contains an open reading frame, in the opposite orientation of prk‐1, potentially encoding 625 amino acids, with similarity to reverse transcriptases. The element is most similar to members of the non‐LTR group of retrotransposable elements. There is weak homology of the predicted amino acid sequence of Rte‐1 to several reverse transcriptase‐like genes identified by the C. elegans genome sequencing consortium, suggesting that there may be a large family of these elements. Southern blots indicate that there are approximately 10–15 additional Rte‐1 elements in the C. elegans Bristol N2 genome and a similar number is found in the genomes of two other geographically distinct strains. The insertion pattern of Rte‐1 is polymorphic between these strains.

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.

Trypanosomes change their transferrin receptor expression to allow effective uptake of host transferrin

In its mammalian host, Trypanosoma brucei covers its iron requirements by receptor‐mediated uptake of host transferrin (Tf). The Tf‐receptor (Tf‐R) is a heterodimeric membrane protein encoded by expression site‐associated gene (ESAG) 6 and 7 located promoter‐proximal in a polycistronic expression site (ES). Each of the 20 ESs encodes a slightly different Tf‐R; these differences strongly affect the binding affinity for Tfs of different hosts. The Tf‐R encoded in the 221 ES has a low affinity for dog Tf. Transfer of trypanosomes with an active 221 ES to dilute dog serum leads to growth arrest, which they can overcome by switching to another ES encoding a Tf‐R with higher affinity for dog Tf. Here we show that trypanosomes can also adapt to dilute dog serum without switching but by replacing the ESAG7 gene in the 221 ES by one from another ES, by deleting ESAG7 from the 221 ES with concomitant upregulation of transcription of ESAG7 in ‘silent’ ESs, by grossly overproducing the 221 Tf‐R or by combinations of these alterations. Our results illustrate the striking genetic flexibility of trypanosomes.

Evidence that J-binding protein 2 is a thymidine hydroxylase catalyzing the first step in the biosynthesis of DNA base J.

The genomic DNA of kinetoplastid parasites contains a unique modified base, beta-d-glucosyl-hydroxymethyluracil or base J. We recently reported that two proteins, called J-binding protein (JBP) 1 and 2, which regulate the levels of J in the genome, display features of the family of Fe(II)-2-oxoglutarate dependent dioxygenases and are likely to be the enzymes catalyzing the first step in J biosynthesis. In this study, we examine the effects of replacing the four conserved residues critical for the activity of this class of enzymes on the function of Leishmania tarentolae JBP2. The results show that each of these four residues is indispensable for the ability of JBP2 to stimulate J synthesis, while mutating non-conserved residues has no consequences. We conclude that JBP2, like JBP1, is in all probability a thymidine hydroxylase involved in the biosynthesis of base J.

Telomeric localization of the modified DNA base J in the genome of the protozoan parasite Leishmania

Base J or β-d-glucosylhydroxymethyluracil is a DNA modification replacing a fraction of thymine in the nuclear DNA of kinetoplastid parasites and of Euglena. J is located in the telomeric sequences of Trypanosoma brucei and in other simple repeat DNA sequences. In addition, J was found in the inactive variant surface glycoprotein (VSG) expression sites, but not in the active expression site of T. brucei, suggesting that J could play a role in transcription silencing in T. brucei. We have now looked at the distribution of J in the genomes of other kinetoplastid parasites. First, we analyzed the DNA sequences immunoprecipitated with a J-antiserum in Leishmania major Friedlin. Second, we investigated the co-migration of J- and telomeric repeat-containing DNA sequences of various kinetoplastids using J-immunoblots and Southern blots of fragmented DNA. We find only ∼1% of J outside the telomeric repeat sequences of Leishmania sp. and Crithidia fasciculata, in contrast to the substantial fraction of non-telomeric J found in T. brucei, Trypanosoma equiperdum and Trypanoplasma borreli. Our results suggest that J is a telomeric base modification, recruited for other (unknown) functions in some kinetoplastids and Euglena.

The structural basis for recognition of base J containing DNA by a novel DNA binding domain in JBP1

The J-binding protein 1 (JBP1) is essential for biosynthesis and maintenance of DNA base-J (β-d-glucosyl-hydroxymethyluracil). Base-J and JBP1 are confined to some pathogenic protozoa and are absent from higher eukaryotes, prokaryotes and viruses. We show that JBP1 recognizes J-containing DNA (J-DNA) through a 160-residue domain, DB-JBP1, with 10 000-fold preference over normal DNA. The crystal structure of DB-JBP1 revealed a helix-turn-helix variant fold, a ‘helical bouquet’ with a ‘ribbon’ helix encompassing the amino acids responsible for DNA binding. Mutation of a single residue (Asp525) in the ribbon helix abrogates specificity toward J-DNA. The same mutation renders JBP1 unable to rescue the targeted deletion of endogenous JBP1 genes in Leishmania and changes its distribution in the nucleus. Based on mutational analysis and hydrogen/deuterium-exchange mass-spectrometry data, a model of JBP1 bound to J-DNA was constructed and validated by small-angle X-ray scattering data. Our results open new possibilities for targeted prevention of J-DNA recognition as a therapeutic intervention for parasitic diseases.

Defining the sequence requirements for the positioning of base J in DNA using SMRT sequencing

Base J (β-D-glucosyl-hydroxymethyluracil) replaces 1% of T in the Leishmania genome and is only found in telomeric repeats (99%) and in regions where transcription starts and stops. This highly restricted distribution must be co-determined by the thymidine hydroxylases (JBP1 and JBP2) that catalyze the initial step in J synthesis. To determine the DNA sequences recognized by JBP1/2, we used SMRT sequencing of DNA segments inserted into plasmids grown in Leishmania tarentolae. We show that SMRT sequencing recognizes base J in DNA. Leishmania DNA segments that normally contain J also picked up J when present in the plasmid, whereas control sequences did not. Even a segment of only 10 telomeric (GGGTTA) repeats was modified in the plasmid. We show that J modification usually occurs at pairs of Ts on opposite DNA strands, separated by 12 nucleotides. Modifications occur near G-rich sequences capable of forming G-quadruplexes and JBP2 is needed, as it does not occur in JBP2-null cells. We propose a model whereby de novo J insertion is mediated by JBP2. JBP1 then binds to J and hydroxylates another T 13 bp downstream (but not upstream) on the complementary strand, allowing JBP1 to maintain existing J following DNA replication.