Trypanosomes have divergent kinesin-2 proteins that function differentially in flagellum biosynthesis and cell viability.

, the causative agent of African sleeping sickness, has a flagellum that is crucial for motility, pathogenicity, and viability. In most eukaryotes, the intraflagellar transport (IFT) machinery drives flagellum biogenesis, and anterograde IFT requires kinesin-2 motor proteins. In this study, we investigated the function of the two kinesin-2 proteins, TbKin2a and TbKin2b, in bloodstream form trypanosomes.

Dynamic localization of SPO11-1 and conformational changes of meiotic axial elements during recombination initiation of maize meiosis.

Meiotic double-strand breaks (DSBs) are generated by the evolutionarily conserved SPO11 complex in the context of chromatin loops that are organized along axial elements (AEs) of chromosomes. However, how DSBs are formed with respect to chromosome axes and the SPO11 complex remains unclear in plants. Here, we confirm that DSB and bivalent formation are defective in maize spo11-1 mutants.

Mad2, Bub3, and Mps1 regulate chromosome segregation and mitotic synchrony in Giardia intestinalis, a binucleate protist lacking an anaphase-promoting complex.

The binucleate pathogen Giardia intestinalis is a highly divergent eukaryote with a semiopen mitosis, lacking an anaphase-promoting complex/cyclosome (APC/C) and many of the mitotic checkpoint complex (MCC) proteins. However, Giardia has some MCC components (Bub3, Mad2, and Mps1) and proteins from the cohesin system (Smc1 and Smc3). Mad2 localizes to the cytoplasm, but Bub3 and Mps1 are either located on chromosomes or in the cytoplasm, depending on the cell cycle stage.

Regulation of cell divisions and differentiation by MALE STERILITY32 is required for anther development in maize.

Male fertility in flowering plants relies on proper division and differentiation of cells in the anther, a process that gives rise to four somatic layers surrounding central germinal cells. The maize gene male sterility32 (ms32) encodes a basic helix-loop-helix (bHLH) transcription factor, which functions as an important regulator of both division and differentiation during anther development. After the four somatic cell layers are generated properly through successive periclinal divisions, in the ms32 mutant, tapetal precursor cells fail to differentiate, and, instead, undergo additional periclinal divisions to form extra layers of cells.

Nutritional control of epigenetic processes in yeast and human cells.

The vitamin folate is required for methionine homeostasis in all organisms. In addition to its role in protein synthesis, methionine is the precursor to S-adenosyl-methionine (SAM), which is used in myriad cellular methylation reactions, including all histone methylation reactions. Here, we demonstrate that folate and methionine deficiency led to reduced methylation of lysine 4 of histone H3 (H3K4) in Saccharomyces cerevisiae.

The Giardia cell cycle progresses independently of the anaphase-promoting complex.

Most cell cycle regulation research has been conducted in model organisms representing a very small part of the eukaryotic domain. The highly divergent human pathogen Giardia intestinalis is ideal for studying the conservation of eukaryotic pathways. Although Giardia has many cell cycle regulatory components, its genome lacks all anaphase-promoting complex (APC) components.

Maize multiple archesporial cells 1 (mac1), an ortholog of rice TDL1A, modulates cell proliferation and identity in early anther development.

To ensure fertility, complex somatic and germinal cell proliferation and differentiation programs must be executed in flowers. Loss-of-function of the maize multiple archesporial cells 1 (mac1) gene increases the meiotically competent population and ablates specification of somatic wall layers in anthers. We report the cloning of mac1, which is the ortholog of rice TDL1A.

Nuclear inheritance and genetic exchange without meiosis in the binucleate parasite Giardia intestinalis.

The protozoan parasite Giardia intestinalis (also known as Giardia lamblia) is a major waterborne pathogen. During its life cycle, Giardia alternates between the actively growing trophozoite, which has two diploid nuclei with low levels of allelic heterozygosity, and the infectious cyst, which has four nuclei and a tough outer wall. Although the formation of the cyst wall has been studied extensively, we still lack basic knowledge about many fundamental aspects of the cyst, including the sources of the four nuclei and their distribution during the transformation from cyst into trophozoite.

RNAi promotes heterochromatic silencing through replication-coupled release of RNA Pol II.

Heterochromatin comprises tightly compacted repetitive regions of eukaryotic chromosomes. The inheritance of heterochromatin through mitosis requires RNA interference (RNAi), which guides histone modification during the DNA replication phase of the cell cycle. Here we show that the alternating arrangement of origins of replication and non-coding RNA in pericentromeric heterochromatin results in competition between transcription and replication in Schizosaccharomyces pombe.

Global transcriptome analysis of two ameiotic1 alleles in maize anthers: defining steps in meiotic entry and progression through prophase I.

Background: Developmental cues to start meiosis occur late in plants. Ameiotic1 (Am1) encodes a plant-specific nuclear protein (AM1) required for meiotic entry and progression through early prophase I. Pollen mother cells (PMCs) remain mitotic in most am1 mutants including am1-489, while am1-praI permits meiotic entry but PMCs arrest at the leptotene/zygotene (L/Z) transition, defining the roles of AM1 protein in two distinct steps of meiosis.

Inna Golubovskaya: the life of a geneticist studying meiosis.

Maize, with its excellent forward genetics and male sterility screens, was used to identify >50 meiotic mutants representing at least 35 genes that affect key prophase processes such as pairing, synapsis, and homologous recombination. Most of these mutants were found by Inna Golubovskaya during the course of her remarkable career as a cytogeneticist. In addition to undertaking general cytological surveys to classify mutant phenotypes, Golubovskaya focused her efforts on characterizing several key regulatory mutants: ameiotic1 (am1), required to establish the meiotic cell cycle in maize; absence of first division (afd1), required for proper prophase chromosome morphology and for meiotic sister-chromatid cohesion leading to a reductive chromosome segregation at the first meiotic division; and plural abnormalities of meiosis (pam1), required for the clustering of telomeres on the nuclear envelope needed for pairing and synapsis.

Coordination of DNA replication and histone modification by the Rik1-Dos2 complex.

Histone modification marks have an important role in many chromatin processes. During DNA replication, both heterochromatin and euchromatin are disrupted ahead of the replication fork and are then reassembled into their original epigenetic states behind the fork. How histone marks are accurately inherited from generation to generation is still poorly understood.

An actin cytoskeleton with evolutionarily conserved functions in the absence of canonical actin-binding proteins.

Giardia intestinalis, a human intestinal parasite and member of what is perhaps the earliest-diverging eukaryotic lineage, contains the most divergent eukaryotic actin identified to date and is the first eukaryote known to lack all canonical actin-binding proteins (ABPs). We sought to investigate the properties and functions of the actin cytoskeleton in Giardia to determine whether Giardia actin (giActin) has reduced or conserved roles in core cellular processes. In vitro polymerization of giActin produced filaments, indicating that this divergent actin is a true filament-forming actin.

Marker reconstitution mutagenesis: a simple and efficient reverse genetic approach.

A novel reverse genetic approach termed 'marker reconstitution mutagenesis' was designed to generate mutational allelic series in genes of interest. This approach consists of two simple steps which utilize two selective markers. First, using one selective marker, a partial fragment of another selective marker gene is inserted adjacently to a gene of interest by homologous recombination.

Rapid tagging and integration of genes in Giardia intestinalis.

We developed a series of plasmids that allow C-terminal tagging of any gene in its endogenous locus in Giardia intestinalis, with different epitope tags (triple hemagglutinin [3HA] and triple Myc [3Myc]) and selection markers (puromycin, neomycin, and a newly developed marker, blasticidin). Using these vectors, cyclin B and aurora kinase were tagged, expressed, and localized.

Ancestral centriole and flagella proteins identified by analysis of Naegleria differentiation.

Naegleria gruberi is a single-celled eukaryote best known for its remarkable ability to form an entire microtubule cytoskeleton de novo during its metamorphosis from an amoeba into a flagellate, including basal bodies (equivalent to centrioles), flagella and a cytoplasmic microtubule array. Our publicly available full-genome transcriptional analysis, performed at 20-minute intervals throughout Naegleria differentiation, reveals vast transcriptional changes, including the differential expression of genes involved in metabolism, signaling and the stress response. Cluster analysis of the transcriptional profiles of predicted cytoskeletal genes reveals a set of 55 genes enriched in centriole components (induced early) and a set of 82 genes enriched in flagella proteins (induced late).

Intermediary metabolism in protists: a sequence-based view of facultative anaerobic metabolism in evolutionarily diverse eukaryotes.

Protists account for the bulk of eukaryotic diversity. Through studies of gene and especially genome sequences the molecular basis for this diversity can be determined. Evident from genome sequencing are examples of versatile metabolism that go far beyond the canonical pathways described for eukaryotes in textbooks.

Maize meiotic mutants with improper or non-homologous synapsis due to problems in pairing or synaptonemal complex formation.

During meiotic prophase homologous chromosomes find each other and pair. Then they synapse, as the linear protein core (axial element or lateral element) of each homologous chromosome is joined together by a transverse central element, forming the tripartite synaptonemal complex (SC). Ten uncloned Zea mays mutants in our collection were surveyed by transmission electron microscopy by making silver-stained spreads of SCs to identify mutants with non-homologous synapsis or improper synapsis.

Naegleria gruberi de novo basal body assembly occurs via stepwise incorporation of conserved proteins.

Centrioles and basal bodies are discrete structures composed of a cylinder of nine microtubule triplets and associated proteins. Metazoan centrioles can be found at mitotic spindle poles and are called basal bodies when used to organize microtubules to form the core structure of flagella. Naegleria gruberi, a unicellular eukaryote, grows as an amoeba that lacks a cytoplasmic microtubule cytoskeleton.

The genome of Naegleria gruberi illuminates early eukaryotic versatility.

Genome sequences of diverse free-living protists are essential for understanding eukaryotic evolution and molecular and cell biology. The free-living amoeboflagellate Naegleria gruberi belongs to a varied and ubiquitous protist clade (Heterolobosea) that diverged from other eukaryotic lineages over a billion years ago. Analysis of the 15,727 protein-coding genes encoded by Naegleria's 41 Mb nuclear genome indicates a capacity for both aerobic respiration and anaerobic metabolism with concomitant hydrogen production, with fundamental implications for the evolution of organelle metabolism.

Interlock formation and coiling of meiotic chromosome axes during synapsis.

The meiotic prophase chromosome has a unique architecture. At the onset of leptotene, the replicated sister chromatids are organized along an axial element. During zygotene, as homologous chromosomes pair and synapse, a synaptonemal complex forms via the assembly of a transverse element between the two axial elements.