William J. Sullivan, Jr.

Permanent URI for this collection

https://hdl.handle.net/1805/42993

Bill Sullivan is the author of Pleased to Meet Me: Genes, Germs, and the Curious Forces That Make Us Who We Are (National Geographic Books), which has been translated into a dozen languages. Sullivan is the Showalter Professor at the Indiana University School of Medicine, where he studies infectious disease. He received his Ph.D. in Cell & Molecular Biology from the University of Pennsylvania and has published over 100 papers in scientific journals. An award-winning researcher, teacher, and science communicator, Sullivan has been featured in a wide variety of outlets, including CNN, Fox & Friends, CBS News, ESPN, The Doctors, New York Post, Wall Street Journal, TEDx, The Scientist, and many more. He has written popular science articles for National Geographic, Discover, Scientific American, Washington Post, WIRED, Psychology Today, The Conversation, and more. He is an editor and writer at PLOS SciComm, chairs the Editorial Advisory Board for ASBMB Today, and serves as a board member of the John Shaw Billings Medical History Society.

Browse

Now showing 1 - 10 of 127

Tagging genes and trapping promoters in Toxoplasma gondii by insertional mutagenesis
(Elsevier, 1997) Roos, David S.; Sullivan, William J., Jr.; Striepen, Boris; Bohne, Wolfgang; Donald, Robert G. K.
Plasmid vectors that incorporate sequence elements from the dehydrofolate reductase-thymidylate synthase (DHFR-TS) locus of Toxoplasma gondii integrate into the parasite genome with remarkably high frequency (>1% of transfected parasites). These vectors may-but need not-include mutant DHFR-TS alleles that confer pyrimethamine resistance to transgenic parasites. Large genomic constructs integrate at the endogenous locus by homologous recombination, but cDNA-derived sequences lacking long stretches of contiguous genomic DNA (due to intron excision) typically integrate into chromosomal DNA by nonhomologous recombination. Nonhomologous integration occurs effectively at random; and coupled with the high frequency of transformation, this allows a large fraction of the parasite genome to be tagged in a single electroporation cuvette. Genomic tagging permits insertional mutagenesis studies conceptually analogous to transposon mutagenesis in bacteria, yeast, Drosophila, etc. In theory (and, thus far, in practice), this allows identification of any gene whose inactivation is not lethal to the haploid tachyzoite form of T. gondii and for which a suitable selection or screen is available. Transformation vectors can be engineered to facilitate rescue of the tagged locus and to include a variety of reporters or selectable markers. Genetic strategies are also possible, using reporters whose function can be assayed by metabolic, visual, or immunological screens to "trap" genes that are activated (or inactivated) under various conditions of interest.
Insertional tagging of at least two loci associated with resistance to adenine arabinoside in Toxoplasma gondii, and cloning of the adenosine kinase locus
(Elsevier, 1999) Sullivan, William J., Jr.; Chiang, Chi-Wu; Wilson, Craig M.; Naguib, Fardos N. M.; el Kouni, Mahmoud H.; Donald, Robert G. K.; Roos, David S.
A genetic approach has been exploited to investigate adenylate salvage pathways in the protozoan parasite Toxoplasma gondii, a purine auxotroph. Using a new insertional mutagenesis vector designed to facilitate the rescue of tagged loci even when multiple plasmids integrate as a tandem array, 15 independent clonal lines resistant to the toxic nucleoside analog adenine arabinoside (AraA) were generated. Approximately two-thirds of these clones lack adenosine kinase (AK) activity. Parallel studies identified an expressed sequence tag (EST) exhibiting a small region of weak similarity to human AK, and this locus was tagged in several AK-deficient insertional mutants. Library screening yielded full-length cDNA and genomic clones. The T. gondii AK gene contains five exons spanning a approximately 3 kb locus, and the predicted coding sequence was employed to identify additional AK genes and cDNAs in the GenBank and dbEST databases. A genomic construct lacking essential coding sequence was used to create defined genetic knock-outs at the T. gondii AK locus, and AK activity was restored using a cDNA-derived minigene. Hybridization analysis of DNA from 13 AraA-resistant insertional mutants reveals three distinct classes: (i) AK-mutants tagged at the AK locus; (ii) AK- mutants not tagged at the AK locus, suggesting the possibility that another locus may be involved in regulating AK expression; and (iii) mutants with normal AK activity (potential transport mutants).
The adenosine transporter of Toxoplasma gondii: Identification by insertional mutagenesis, cloning, and recombinant expression
(Elsevier, 1999) Chiang, Chi-Wu; Carter, Nicola; Sullivan, William J., Jr.; Donald, Robert G. K.; Roos, David S.; Naguib, Fardos N. M.; el Kouni, Mahmoud H.; Ullman, Buddy; Wilson, Craig M.
Purine transport into the protozoan parasite Toxoplasma gondii plays an indispensable nutritional function for this pathogen. To facilitate genetic and biochemical characterization of the adenosine transporter of the parasite, T. gondii tachyzoites were transfected with an insertional mutagenesis vector, and clonal mutants were selected for resistance to the cytotoxic adenosine analog adenine arabinoside (Ara-A). Whereas some Ara-A-resistant clones exhibited disruption of the adenosine kinase (AK) locus, others displayed normal AK activity, suggesting that a second locus had been tagged by the insertional mutagenesis plasmid. These Ara-A(r) AK+ mutants displayed reduced adenosine uptake capability, implying a defect in adenosine transport. Sequences flanking the transgene integration point in one mutant were rescued from a genomic library of Ara-A(r) AK+ DNA, and Southern blot analysis revealed that all Ara-A(r) AK+ mutants were disrupted at the same locus. Probes derived from this locus, designated TgAT, were employed to isolate genomic and cDNA clones from wild-type libraries. Conceptual translation of the TgAT cDNA open reading frame predicts a 462 amino acid protein containing 11 transmembrane domains, a primary structure and membrane topology similar to members of the mammalian equilibrative nucleoside transporter family. Expression of TgAT cRNA in Xenopus laevis oocytes increased adenosine uptake capacity in a saturable manner, with an apparent K(m) value of 114 microM. Uptake was inhibited by various nucleosides, nucleoside analogs, hypoxanthine, guanine, and dipyridamole. The combination of genetic and biochemical studies demonstrates that TgAT is the sole functional adenosine transporter in T. gondii and a rational target for therapeutic intervention.
Recombinant expression, purification, and characterization of Toxoplasma gondii adenosine kinase
(Elsevier, 1999) Darling, John A.; Sullivan, William J., Jr.; Carter, Darrick; Ullman, Buddy; Roos, David S.
Toxoplasma gondii lacks the capacity to synthesize purines de novo, and adenosine kinase (AK)-mediated phosphorylation of salvaged adenosine provides the major route of purine acquisition by this parasite. T. gondii AK thus represents a promising target for rational design of antiparasitic compounds. In order to further our understanding of this therapeutically relevant enzyme, an AK cDNA from T. gondii was overexpressed in E. coli using the pBAce expression system, and the recombinant protein was purified to apparent homogeneity using conventional protein purification techniques. Kinetic analysis of TgAK revealed Km values of 1.9 microM for adenosine and 54.4 microM for ATP, with a k(cat) of 26.1 min(-1). Other naturally occurring purine nucleosides, nucleobases, and ribose did not significantly inhibit adenosine phosphorylation, but inhibition was observed using certain purine nucleoside analogs. Adenine arabinoside (AraA), 4-nitrobenzylthioinosine (NBMPR), and 7-deazaadenosine (tubercidin) were all shown to be substrates of T. gondii AK. Transgenic AK knock-out parasites were resistant to these compounds in cell culture assays, consistent with their proposed action as subversive substrates in vivo.
Parasite-specific eIF2 (eukaryotic initiation factor-2) kinase required for stress-induced translation control
(Portland Press, 2004) Sullivan, William J., Jr.; Narasimhan, Jana; Bhatti, Micah M.; Wek, Ronald C.; Pharmacology and Toxicology, School of Medicine
The ubiquitous intracellular parasite Toxoplasma gondii (phylum Apicomplexa) differentiates into an encysted form (bradyzoite) that can repeatedly re-emerge as a life-threatening acute infection (tachyzoite) upon impairment of immunity. Since the switch from tachyzoite to bradyzoite is a stress-induced response, we sought to identify components related to the phosphorylation of the alpha subunit of eIF2 (eukaryotic initiation factor-2), a well-characterized event associated with stress remediation in other eukaryotic systems. In addition to characterizing Toxoplasma eIF2alpha (TgIF2alpha), we have discovered a novel eIF2 protein kinase, designated TgIF2K-A (Toxoplasma gondii initiation factor-2kinase). Although the catalytic domain of TgIF2K-A contains sequence and structural features that are conserved among members of the eIF2 kinase family, TgIF2K-A has an extended N-terminal region that is highly divergent from other eIF2 kinases. TgIF2K-A specifically phosphorylates the regulatory serine residue of yeast eIF2alpha in vitro and in vivo, and can modulate translation when expressed in the yeast model system. We also demonstrate that TgIF2K-A phosphorylates the analogous regulatory serine residue of recombinant TgIF2alpha in vitro. Finally, we demonstrate that TgIF2alpha phosphorylation in tachyzoites is enhanced in response to heat shock or alkaline stress, conditions known to induce parasite differentiation in vitro. Collectively, this study suggests that eIF2 kinase-mediated stress responses are conserved in Apicomplexa, and a novel family member exists that may control parasite-specific events, including the clinically relevant conversion into bradyzoite cysts.
Purine Salvage Pathways in the Apicomplexan Parasite Toxoplasma gondii
(Elsevier, 2004) Chaudhary, Kshitiz; Darling, John A.; Fohl, Leah M.; Sullivan, William J., Jr.; Donald, Robert G. K.; Pfefferkorn, Elmer R.; Ullman, Buddy; Roos, David S.; Pharmacology and Toxicology, School of Medicine
We have exploited a variety of molecular genetic, biochemical, and genomic techniques to investigate the roles of purine salvage enzymes in the protozoan parasite Toxoplasma gondii. The ability to generate defined genetic knockouts and target transgenes to specific loci demonstrates that T. gondii uses two (and only two) pathways for purine salvage, defined by the enzymes hypoxanthine-xanthine-guanine phosphoribosyltransferase (HXGPRT) and adenosine kinase (AK). Both HXGPRT and AK are single-copy genes, and either one can be deleted, indicating that either one of these pathways is sufficient to meet parasite purine requirements. Fitness defects suggest both pathways are important for the parasite, however, and that the salvage of adenosine is more important than salvage of hypoxanthine and other purine nucleobases. HXGPRT and AK cannot be deleted simultaneously unless one of these enzymes is provided in trans, indicating that alternative routes of functionally significant purine salvage are lacking. Despite previous reports to the contrary, we found no evidence of adenine phosphoribosyltransferase (APRT) activity when parasites were propagated in APRT-deficient host cells, and no APRT ortholog is evident in the T. gondii genome. Expression of Leishmania donovani APRT in transgenic T. gondii parasites yielded low levels of activity but did not permit genetic deletion of both HXGPRT and AK. A detailed comparative genomic study of the purine salvage pathway in various apicomplexan species highlights important differences among these parasites.
Histone-Modifying Complexes Regulate Gene Expression Pertinent to the Differentiation of the Protozoan Parasite Toxoplasma gondii
(Taylor & Francis, 2005) Saksouk, Nehmé; Bhatti, Micah M.; Kieffer, Sylvie; Smith, Aaron T.; Musset, Karine; Garin, Jérôme; Sullivan, William J., Jr.; Cesbron-Delauw, Marie-France; Hakimi, Mohamed-Ali; Pharmacology and Toxicology, School of Medicine
Pathogenic apicomplexan parasites like Toxoplasma and Plasmodium (malaria) have complex life cycles consisting of multiple stages. The ability to differentiate from one stage to another requires dramatic transcriptional changes, yet there is a paucity of transcription factors in these protozoa. In contrast, we show here that Toxoplasma possesses extensive chromatin remodeling machinery that modulates gene expression relevant to differentiation. We find that, as in other eukaryotes, histone acetylation and arginine methylation are marks of gene activation in Toxoplasma. We have identified mediators of these histone modifications, as well as a histone deacetylase (HDAC), and correlate their presence at target promoters in a stage-specific manner. We purified the first HDAC complex from apicomplexans, which contains novel components in addition to others previously reported in eukaryotes. A Toxoplasma orthologue of the arginine methyltransferase CARM1 appears to work in concert with the acetylase TgGCN5, which exhibits an unusual bias for H3 [K18] in vitro. Inhibition of TgCARM1 induces differentiation, showing that the parasite life cycle can be manipulated by interfering with epigenetic machinery. This may lead to new approaches for therapy against protozoal diseases and highlights Toxoplasma as an informative model to study the evolution of epigenetics in eukaryotic cells.
The protozoan parasite Cryptosporidium parvum possesses two functionally and evolutionarily divergent replication protein A large subunits
(Elsevier, 2005) Rider, S. Dean, Jr.; Cai, Xiaomin; Sullivan, William J., Jr.; Smith, Aaron T.; Radke, Jay; White, Michael; Zhu, Guan; Pharmacology and Toxicology, School of Medicine
Very little is known about protozoan replication protein A (RPA), a heterotrimeric complex critical for DNA replication and repair. We have discovered that in medically and economically important apicomplexan parasites, two unique RPA complexes may exist based on two different types of large subunit RPA1. In this study, we characterized the single-stranded DNA binding features of two distinct types (i.e. short and long) of RPA1 subunits from Cryptosporidium parvum (CpRPA1A and CpRPA1B). These two proteins differ from human RPA1 in their intrinsic single-stranded DNA binding affinity (K) and have significantly lower cooperativity (omega). We also identified the RPA2 and RPA3 subunits from C. parvum, the latter of which had yet to be reported to exist in any protozoan. Using fluorescence resonance energy transfer technology and pull-down assays, we confirmed that these two subunits interact with each other and with CpRPA1A and CpRPA1B. This suggests that the heterotrimeric structure of RPA complexes may be universally conserved from lower to higher eukaryotes. Bioinformatic analyses indicate that multiple types of RPA1 are present in the other apicomplexans Plasmodium and Toxoplasma. Apicomplexan RPA1 proteins are phylogenetically more related to plant homologues and probably arose from a single gene duplication event prior to the expansion of the apicomplexan lineage. Differential expression during the life cycle stages in three apicomplexan parasites suggests that the two RPA1 types exercise specialized biological functions.
MYST Family Histone Acetyltransferases in the Protozoan Parasite Toxoplasma gondii
(American Society for Microbiology, 2005) Smith, Aaron T.; Tucker-Samaras, Samantha D.; Fairlamb, Alan H.; Sullivan, William J., Jr.; Pharmacology and Toxicology, School of Medicine
The restructuring of chromatin precedes tightly regulated events such as DNA transcription, replication, and repair. One type of chromatin remodeling involves the covalent modification of nucleosomes by histone acetyltransferase (HAT) complexes. The observation that apicidin exerts antiprotozoal activity by targeting a histone deacetyltransferase has prompted our search for more components of the histone modifying machinery in parasitic protozoa. We have previously identified GNAT family HATs in the opportunistic pathogen Toxoplasma gondii and now describe the first MYST (named for members MOZ, Ybf2/Sas3, Sas2, and Tip60) family HATs in apicomplexa (TgMYST-A and -B). The TgMYST-A genomic locus is singular and generates a approximately 3.5-kb transcript that can encode two proteins of 411 or 471 amino acids. TgMYST-B mRNA is approximately 7.0 kb and encodes a second MYST homologue. In addition to the canonical MYST HAT catalytic domain, both TgMYST-A and -B possess an atypical C2HC zinc finger and a chromodomain. Recombinant TgMYST-A exhibits a predilection to acetylate histone H4 in vitro at lysines 5, 8, 12, and 16. Antibody generated to TgMYST-A reveals that both the long and short (predominant) versions are present in the nucleus and are also plentiful in the cytoplasm. Moreover, both TgMYST-A forms are far more abundant in rapidly replicating parasites (tachyzoites) than encysted parasites (bradyzoites). A bioinformatics survey of the Toxoplasma genome reveals numerous homologues known to operate in native MYST complexes. The characterization of TgMYST HATs represents another important step toward understanding the regulation of gene expression in pathogenic protozoa and provides evolutionary insight into how these processes operate in eukaryotic cells in general.
Histone Acetylase GCN5 Enters the Nucleus via Importin-α in Protozoan Parasite Toxoplasma
(Elsevier, 2005) Bhatti, Micah M.; Sullivan, William J., Jr.; Pharmacology and Toxicology, School of Medicine
The histone acetyltransferase GCN5 acetylates nucleosomal histones to alter gene expression. How GCN5 gains entry into the nucleus of the cell has not been determined. We have mapped a six-amino acid motif (RKRVKR) that serves as a necessary and sufficient nuclear localization signal (NLS) for GCN5 in the protozoan pathogen Toxoplasma gondii (TgGCN5). Virtually nothing is known about nucleocytoplasmic transport in these parasites (phylum Apicomplexa), and this study marks the first demonstrated NLS delineated for members of the phylum. The TgGCN5 NLS has predictive value because it successfully identifies other nuclear proteins in three different apicomplexan genomic databases. Given the basic composition of the T. gondii NLS, we hypothesized that TgGCN5 physically interacts with importin-alpha, the main transport receptor in the importin/karyopherin nuclear import pathway. We cloned the importin-alpha gene from T. gondii (TgIMPalpha), which encodes a protein of 545 amino acids that possesses an importin-beta-binding domain and armadillo/beta-catenin-like repeats. In vitro co-immunoprecipitation experiments confirm that TgIMPalpha directly interacts with TgGCN5, but this interaction is abolished if the TgGCN5 NLS is deleted. Taken together, these data argue that TgGCN5 gains access to the parasite nucleus by interacting with TgIMPalpha. Bioinformatics analysis of the T. gondii genome reveals that other components of the importin pathway are present in the organism. This study demonstrates the utility of T. gondii as a model for the study of nucleocytoplasmic trafficking in early eukaryotic cells.

Browse

Browsing William J. Sullivan, Jr. by Issue Date

Results Per Page

Sort Options