Giardia lamblia Nek1 and Nek2 kinases affect mitosis and excystation.

The NIMA-related serine/threonine kinases (Neks) function in the cell cycle and regulate ciliary and flagellar length. The Giardia lamblia genome encodes 198 Neks, of which 56 are predicted to be active. Here we believe that we report the first functional analysis of two G. lamblia Neks. The GlNek1 and GlNek2 kinase domains share 57% and 43% identity to the kinase domains of human Nek1 and Nek2, respectively. Both GlNeks are active in vitro, have dynamic relocalisation during the cell cycle, and are expressed throughout the life cycle, with GlNek1 being upregulated in cysts. Over-expression of inactive GlNek1 delays disassembly of the parental attachment disc and cytokinesis, whilst over-expression of either wild type GlNek1 or inactive mutant GlNek2 inhibits excystation.

Mining the Giardia genome and proteome for conserved and unique basal body proteins.

Giardia lamblia is a flagellated protozoan parasite and a major cause of diarrhoea in humans. Its microtubular cytoskeleton mediates trophozoite motility, attachment and cytokinesis, and is characterised by an attachment disk and eight flagella that are each nucleated in a basal body. To date, only 10 giardial basal body proteins have been identified, including universal signalling proteins that are important for regulating mitosis or differentiation. In this study, we have exploited bioinformatics and proteomic approaches to identify new Giardia basal body proteins and confocal microscopy to confirm their localisation in interphase trophozoites. This approach identified 75 homologs of conserved basal body proteins in the genome including 65 not previously known to be associated with Giardia basal bodies. Thirteen proteins were confirmed to co-localise with centrin to the Giardia basal bodies. We also demonstrate that most basal body proteins localise to additional cytoskeletal structures in interphase trophozoites. This might help to explain the roles of the four pairs of flagella and Giardia-specific organelles in motility and differentiation. A deeper understanding of the composition of the Giardia basal bodies will contribute insights into the complex signalling pathways that regulate its unique cytoskeleton and the biological divergence of these conserved organelles.

Novel core promoter elements and a cognate transcription factor in the divergent unicellular eukaryote Trichomonas vaginalis.

A highly conserved DNA initiator (Inr) element has been the only core promoter element described in the divergent unicellular eukaryote Trichomonas vaginalis, although genome analyses reveal that only ∼75% of protein-coding genes appear to contain an Inr. In search of another core promoter element(s), a nonredundant database containing 5' untranslated regions of expressed T. vaginalis genes was searched for overrepresented DNA motifs and known eukaryotic core promoter elements. In addition to identifying the Inr, two elements that lack sequence similarity to the known protein-coding gene core promoter, motif 3 (M3) and motif 5 (M5), were identified. Mutational and functional analyses demonstrate that both are novel core promoter elements. M3 [(A/G/T)(A/G)C(G/C)G(T/C)T(T/A/G)] resembles a Myb recognition element (MRE) and is bound specifically by a unique protein with a Myb-like DNA binding domain. The M5 element (CCTTT) overlaps the transcription start site and replaces the Inr as an alternative, gene-specific initiator element. Transcription specifically initiates at the second cytosine within M5, in contrast to characteristic initiation by RNA polymerase II at an adenosine. In promoters that combine M3 with either M5 or Inr, transcription initiation is regulated by the M3 motif.

Draft genome sequence of the sexually transmitted pathogen Trichomonas vaginalis.

We describe the genome sequence of the protist Trichomonas vaginalis, a sexually transmitted human pathogen. Repeats and transposable elements comprise about two-thirds of the approximately 160-megabase genome, reflecting a recent massive expansion of genetic material. This expansion, in conjunction with the shaping of metabolic pathways that likely transpired through lateral gene transfer from bacteria, and amplification of specific gene families implicated in pathogenesis and phagocytosis of host proteins may exemplify adaptations of the parasite during its transition to a urogenital environment. The genome sequence predicts previously unknown functions for the hydrogenosome, which support a common evolutionary origin of this unusual organelle with mitochondria.

Trichomonas vaginalis initiator binding protein (IBP39) and RNA polymerase II large subunit carboxy terminal domain interaction.

The core promoter that directs RNA polymerase to the start of transcription in the protist Trichomonas vaginalis is an initiator (Inr) element recognized by the Inr Binding Protein, IBP39. This nuclear protein is composed of two domains: a 14.5 kDa amino (N-terminal) and a 25 kDa carboxy terminal domain (C-domain). Here we describe the identification of an IBP39-interacting protein by screening a T. vaginalis expression library using a two-hybrid system with the IBP39 C-domain as bait. The CTD of the large subunit of RNAP II was found to specifically interact with the C-domain. The specificity and nature of the interaction between the CTD of RNAP II and the C-domain of IBP39 was validated by three independent biochemical methods: co-immunoprecipitation with epitope-tagged proteins, affinity chromatography and enzyme linked ligand sorbent (ELLSA) assays. Binding was shown to involve hydrophobic bonds and to have a disassociation constant (K(d)) of 690 nM (+/-55). These results confirm and extend our previous binding studies using a peptide composed of the last nine amino acids of RNAP II CTD [Schumacher MA, Lau AOT, Johnson PJ. Structural basis of core promoter recognition in a primitive eukaryote. Cell 2003;115:413-24] that predicted an interaction between the CTD and IBP39. These data further demonstrate that IBP39 minimally possesses two functional domains: a N-terminal DNA binding domain (that recognizes the Inr) [Liston DR, Johnson PJ. Analysis of a ubiquitous promoter element in a primitive eukaryote: early evolution of the initiator element. Mol Cell Biol 1999;19:2380-8] and a C-terminal protein binding domain that recognizes the RNAP II CTD, an interaction that may be critical for recruiting RNAP II for initiation of transcription.