Deshmukh Gopaul

Institut Pasteur · Department of Education

Diseases caused by apicomplexan parasites, such as malaria and toxoplasmosis cause ∼200 million (worldwide) and 1 million (Europe) infections, respectively, every year. Apicomplexa possess a non-photosynthetic organelle homologous to the plant chloroplast, the so-called apicoplast, that is essential for their growth and survival. This study focused...

Thio- and seleno-α,β- and α,β,γ-CNA (Constrained Nucleic Acid) dinucleotides in which two or three torsional angles of the sugar/phosphate backbone are controlled within a dioxaphosphorinane structure have been prepared. Their structural determination have been carried out by means of NMR to show only slight variation on the torsional angle control...

Genetic variation is an essential means of evolution and adaptation in many organisms in response to environmental change. Certain DNA alterations can be carried out by site-specific recombinases (SSRs) that fall into two families: the serine and the tyrosine recombinases. SSRs are seldom found in eukaryotes. A gene homologous to a tyrosine site-sp...

Oligonucleotides used for qRT-PCR, cloning and EMSA assays. The DNA primers used for cloning and qRT-PCR were single stranded, whereas the ones destined for EMSAs were annealed to their reverse complement to form the corresponding double stranded DNA. The EMSA DNAs, when specified, were uniquely 5′ labeled with DY682 on the strands specified in thi...

Expression profile for Pf-Int during life stages of P. falciparum. A) mRNA expression of Pf-Int was examined by real-time PCR at the three parasite living stages: ring, trophozoite and schizont. Values are the mean of two independent experiments whose standard deviation is shown by the bars. Values are expressed as relative to the m-RNA level at ri...

Amino acid sequence alignment of Pf-Int with its homologous. Clustal alignment of Pf-Int with its homologues present in A) P. vivax, P. chabaudii, P. bergheii, P. knowlesi and P. yoelii, and B) Toxoplasma gondii and Neospora caninum. (TIF)

Pf-Int knock-out strategy and its validation. The Pf-Int gene was knocked out by a double cross over reaction in the 3D7 line. A) The Pf-Int-KO parasite was created using the plasmid pCC1-Pf-Int and WR99210 and ganciclovir selection. In pCC1-Pf-Int plasmid, the hDHFR resistance cassette (cyan) was inserted between two regions of the Pf-Int gene: Pf...

Analysis of DNA binding by Pf-Int. A) Supershift assay confirms the presence of Pf-Int bound to the DNA. The binding of His6-Pf-Int-C192 to Selex8 DNA target was examined by supershift using either an antibody raised against the purified recombinant Pf-Int or a commercial anti-His5 in combination with a His-tagged version of Pf-Int-C192 protein. 10...

Analysis of DNA binding by Pf-Int. A) The presence of 900 mM NaCl in the binding buffer of Pf-Int-C192/Selex8 was also tested. B) 10 nM of labeled fragment 1 gDNA target were allowed to bind with increasing amounts of Pf-Int-C162 (lanes 1 to 10). 10 nM of labeled fragment 1 gDNA target were incubated with 3 µM Pf-Int-C162 for 20 minutes (Lane 12) a...

Potential DNA targets identified for Pf-Int. A) Targets identified by sequencing of eluted fragments retained by Pf-Int. B) Targets identified by chip hybridization of eluted fragments. (DOCX)

The Mycobacterium tuberculosis PhoP/PhoR two-component signal transduction system controls the expression of about 2% of the genome and plays a major role in pathogenicity. However, its regulon has not been well characterized. The binding site of PhoP transcription regulator was identified in the upstream regions of msl3, pks2, lipF and fadD21 gene...

Electrophoretic mobility assays with the lipFb, lipFc, msl3b, lipFa2 and pks2c fragments and PhoP-P. lipFb (227 bp), lipFc (182 bp), msl3b (98 bp), lipFa2 (89 bp) and pks2c (77 bp) fragments were incubated with PhoP-P in the presence of poly dI-dC at 10 µg/ml and run on a native polyacrylamide gel (8%), in 0.5× TBE buffer. Each fragment was incubat...

Electrophoretic mobility assays for the lipFa, pks2, msl3 and fadD21 fragments with phosphorylated (P) and unphosphorylated (NP) PhoP. The large DNA fragments initially selected — lipFa (222 bp), pks2 (148 bp), msl3a (235 bp), and fadD21 (210 bp) — were incubated with PhoP-P and unphosphorylated PhoP in the presence of poly dI-dC at 10 µg/ml and ru...

List of genes with DR1, DR2 and DR3 in their upstream regions according to Figure 5. First Excel spreadsheet: DR1+DR2; Second spreadsheet: DR1+DR2+DR3. (XLS)

Integrons are able to incorporate exogenous genes embedded in mobile cassettes, by a site-specific recombination mechanism. Gene cassettes are collected at the attI site, via an integrase mediated recombination between the cassette recombination site, attC, and the attI site. Interestingly, only three nucleotides are conserved between attC and attI...

Similarities between Mycobacterium tuberculosis phoP-phoR mutants and the attenuated laboratory strain M. tuberculosis H37Ra in terms of morphological and cytochemical properties, lipid content, gene expression and virulence attenuation prompted us to analyze the functionality of this two-component regulator in the latter strain. Sequence analysis...

The integron platform codes for an integrase (IntI) from the tyrosine family of recombinases that mediates recombination between a proximal double-strand recombination site, attI and a single-strand target recombination site, attC. The attI site is only recognized by its cognate integrase, while the various tested attCs sites are recombined by seve...

Lateral DNA transfer--the movement of genetic traits between bacteria--has a profound impact on genomic evolution and speciation. The efficiency with which bacteria incorporate genetic information reflects their capacity to adapt to changing environmental conditions. Integron integrases are proteins that mediate site-specific DNA recombination betw...

Abstract Cre recombinase is a member of a large family of site-specific recombination enzymes that performs a cut-and-paste operation between two specific DNA sequences. Our goal has been to understand the mechanism of this complex reaction by trapping and characterizing the three-dimensional structures of each of the reaction intermediates. This w...

Cre recombinase catalyzes site-specific recombination between two 34-bp loxP sites in a variety of DNA substrates. At the start of the recombination pathway, the loxP sites are each bound by two recombinase molecules, and synapsis of the sites is mediated by Cre-Cre interactions. We describe the structures of synaptic complexes formed between a sym...

Three-dimensional structural information on the integrase family of site-specific recombinases has only recently become available, with the crystal structures of catalytic domains, full-length proteins and protein-DNA complexes of this family reported over the past two years. These results have led to a model for the overall architecture and active...

We have determined the X-ray crystal structures of two DNA Holliday junctions (HJs) bound by Cre recombinase. The HJ is a four-way branched structure that occurs as an intermediate in genetic recombination pathways, including site-specific recombination by the lambda-integrase family. Cre recombinase is an integrase family member that recombines 34...

During site-specific DNA recombination, which brings about genetic rearrangement in processes such as viral integration and excision and chromosomal segregation, recombinase enzymes recognize specific DNA sequences and catalyse the reciprocal exchange of DNA strands between these sites. The bacteriophage recombinase Cre catalyses site-specific reco...

Protozoa depend on purine salvage for nucleic acid synthesis. An abundant salvage enzyme in Crithidia fasciculata is the inosine-uridine nucleoside hydrolase (IU-nucleoside hydrolase). The enzyme was cloned by polymerase chain reaction techniques using primers corresponding to the amino acid sequences of tryptic fragments and to the miniexon of C....

Protozoan parasites rely on the host for purines since they lack a de novo synthetic pathway. Crithidia fasciculata salvages exogenous inosine primarily through hydrolysis of the N-ribosidic bond using several nucleoside hydrolases. The most abundant nucleoside hydrolase is relatively nonspecific but prefers inosine and uridine as substrates. Here...

The structure of rat intestinal fatty acid binding protein (I-FABP) with bound oleate (C18:1) has been refined with x-ray diffraction data to a resolution of 1.75 A. The protein contains 10 anti-parallel beta strands composed of 99 residues and 2 short helices of 14 residues. Oleate is located in the interior of the protein in a bent conformation w...

The structure of rat intestinal fatty acid binding protein (I-FABP) with bound oleate (C18:l) has been refinTd with x-ray diffraction data to a resolution of 1.75 A. The protein contains 10 anti-parallel j3 strands composed of 99 residues and 2 short helices of 14 residues. Oleate is located in the interior of the protein in a bent conformation wit...