Two thymidine hydroxylases differentially regulate the formation of glucosylated DNA at regions flanking polymerase II polycistronic transcription units throughout the genome of Trypanosoma brucei.

Base J is a hypermodified DNA base localized primarily to telomeric regions of the genome of Trypanosoma brucei. We have previously characterized two thymidine-hydroxylases (TH), JBP1 and JBP2, which regulate J-biosynthesis. JBP2 is a chromatin re-modeling protein that induces de novo J-synthesis, allowing JBP1, a J-DNA binding protein, to stimulate additional J-synthesis. Here, we show that both JBP2 and JBP1 are capable of stimulating de novo J-synthesis. We localized the JBP1- and JBP2-stimulated J by anti-J immunoprecipitation and high-throughput sequencing. This genome-wide analysis revealed an enrichment of base J at regions flanking polymerase II polycistronic transcription units (Pol II PTUs) throughout the T. brucei genome. Chromosome-internal J deposition is primarily mediated by JBP1, whereas JBP2-stimulated J deposition at the telomeric regions. However, the maintenance of J at JBP1-specific regions is dependent on JBP2 SWI/SNF and TH activity. That similar regions of Leishmania major also contain base J highlights the functional importance of the modified base at Pol II PTUs within members of the kinetoplastid family. The regulation of J synthesis/localization by two THs and potential biological function of J in regulating kinetoplastid gene expression is discussed.

JBP1 and JBP2 are two distinct thymidine hydroxylases involved in J biosynthesis in genomic DNA of African trypanosomes.

Genomic DNA of African trypanosomes contains a hypermodified thymidine residue termed base J (beta-d-glucosyl-HOMedU). This modified base is localized primarily to repetitive DNA, namely the telomeres, and is implicated in the regulation of antigenic variation. The base is synthesized in a two-step pathway. Initially, a thymidine residue in DNA is hydroxylated by a thymidine hydroxylase (TH). This intermediate (HOMedU) is then glucosylated to form base J. Two proteins involved in J synthesis, JBP1 (J binding protein 1) and JBP2, contain a putative TH domain related to the family of Fe(2+)/2-oxoglutarate-dependent hydroxylases. We have previously shown that mutations in the TH domain of JBP1 kill its ability to stimulate J synthesis. Here we show that mutation of key residues in the TH domain of JBP2 ablate its ability to induce de novo J synthesis. While the individual JBP1 null and JBP2 null trypanosomes have reduced J levels, the deletion of both JBP1 and JBP2 generates a cell line that completely lacks base J but still contains glucosyl-transferase activity. Reintroduction of JBP2 in the J-null trypanosome stimulates HOMedU formation and site-specific synthesis of base J. We conclude that JBP2 and JBP1 are the TH enzymes involved in J biosynthesis.

In vitro excystation of metacercariae of Microphallus turgidus under different physicochemical conditions.

This study examined the excystation of metacercarial cysts of Microphallus turgidus under varying conditions of temperature, NaCl concentration, and pH. Metacercarial cysts were dissected from abdominal muscle tissue of grass shrimp Palaemonetes pugio, and immediately incubated for 24 h under one of the following abiotic conditions: temperature (22, 26, 30, 34, 38, and 42 degrees C), NaCl concentrations (0.5, 0.6, 0.7, 0.8, and 0.9%), pH (2, 3, 4, 5, 6, 7, and 8), and percent excystation was recorded. The optimum in vitro excystation of metacercarial cysts occurred in 0.7% saline at 38 degrees C at pH 7; 92% of the cysts excysted under these conditions. Our results indicate that such a combination of extrinsic factors is provided by the definitive host for successful excystation of M. turgidus.