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

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

The APC/C recruits cyclin B1–Cdk1–Cks in prometaphase before D box recognition to control mitotic exit

Prior associations with the APC/C complex during prometaphase makes cyclin B1 a better substrate for the cell cycle–regulating ubiquitin ligase in metaphase (see also a related paper by Di Fiore et al. in this issue)

Southern blots and J-immunoblots of genomic DNA of and (bloodstream form) digested with a variety of frequently cutting restriction enzymes

<p><b>Copyright information:</b></p><p>Taken from "Telomeric localization of the modified DNA base J in the genome of the protozoan parasite "</p><p></p><p>Nucleic Acids Research 2007;35(7):2116-2124.</p><p>Published online 28 Feb 2007</p><p>PMCID:PMC1874636.</p><p>© 2007 The Author(s)</p> The DNA was treated as in . The blot was incubated with the J-antisera (α-J) followed by hybridization with a telomeric probe (telo). () Southern blot of genomic DNA of . () Southern blot of genomic DNA of (bloodstream form). The bands migrating at the top of the lanes 1, 2 and 4 that strongly react with the J-antisera are probably due to the 50 bp repeats as these are digested by RsaI (which was used in the lanes 3, 5, 6 and 7). Lanes 1. AluI, HpaII, BsrGI, HaeII; 2. AluI, HpaII, Sau3AI, TaqI; 3. AluI, HpaII, CfoI, RsaI; 4. BsrGI, HaeII, Sau3AI, Taq I; 5. BsrGI, HaeII, CfoI, RsaI; 6. Sau3AI, RsaI, CfoI, TaqI; 7. AluI, HpaII, BsrGI, HaeII, Sau3AI, RsaI, CfoI, TaqI. ND stands for not digested

Southern blot and J-immunoblot of genomic DNA of various kinetoplastid parasites digested with frequently cutting restriction enzymes

<p><b>Copyright information:</b></p><p>Taken from "Telomeric localization of the modified DNA base J in the genome of the protozoan parasite "</p><p></p><p>Nucleic Acids Research 2007;35(7):2116-2124.</p><p>Published online 28 Feb 2007</p><p>PMCID:PMC1874636.</p><p>© 2007 The Author(s)</p> DNA was digested with the restriction enzymes AluI, AvaII, CfoI, HinfI, RsaI, SspI, size-fractionated and blotted as described in . The left panel shows the result after incubation with the J-antiserum (α-J). The right panel shows the result of the hybridization with the telomeric probe (telo)

Southern blot and J-immunoblot of genomic DNA of Friedlin, and digested with frequently cutting restriction enzymes

<p><b>Copyright information:</b></p><p>Taken from "Telomeric localization of the modified DNA base J in the genome of the protozoan parasite "</p><p></p><p>Nucleic Acids Research 2007;35(7):2116-2124.</p><p>Published online 28 Feb 2007</p><p>PMCID:PMC1874636.</p><p>© 2007 The Author(s)</p> () Genomic DNA of Friedlin was digested with the enzymes AluI, BsrGI, BstUI, CfoI, HaeII, HpaII, Sau3AI and TaqI and size-fractionated by electrophoresis in an agarose gel and blotted on a nylon membrane. The blot was incubated with the J-antiserum (α-J) and after analysis, hybridized with telomeric (telo) and LST-RA radioactively labeled DNA oligonucleotide probes. () Southern blot and J-immunoblot of genomic DNA of , , and . DNA of was digested with AluI, AvaII, CfoI, HinfI, RsaI, SspI. DNA of and was digested with the enzymes listed in A. The combination of restriction enzymes was optimized to digest the greatest variety of DNA repetitive sequences. The left panel shows the result after incubation with the J-antiserum (α-J). The right panel shows the result of the hybridization with the telomeric probe (telo)