Depletion of UBC9 Causes Nuclear Defects during the Vegetative and Sexual Life Cycles in Tetrahymena thermophila.

Ubc9p is the sole E2-conjugating enzyme for SUMOylation, and its proper function is required for regulating key nuclear events such as transcription, DNA repair, and mitosis. In Tetrahymena thermophila, the genome is separated into a diploid germ line micronucleus (MIC) that divides by mitosis and a polyploid somatic macronucleus (MAC) that divides amitotically. This unusual nuclear organization provides novel opportunities for the study of SUMOylation and Ubc9p function.

SUMOylation is developmentally regulated and required for cell pairing during conjugation in Tetrahymena thermophila.

The covalent attachment of small ubiquitin-like modifier (SUMO) to target proteins regulates numerous nuclear events in eukaryotes, including transcription, mitosis and meiosis, and DNA repair. Despite extensive interest in nuclear pathways within the field of ciliate molecular biology, there have been no investigations of the SUMO pathway in Tetrahymena. The developmental program of sexual reproduction of this organism includes cell pairing, micronuclear meiosis, and the formation of a new somatic macronucleus.

Highly precise and developmentally programmed genome assembly in Paramecium requires ligase IV-dependent end joining.

During the sexual cycle of the ciliate Paramecium, assembly of the somatic genome includes the precise excision of tens of thousands of short, non-coding germline sequences (Internal Eliminated Sequences or IESs), each one flanked by two TA dinucleotides. It has been reported previously that these genome rearrangements are initiated by the introduction of developmentally programmed DNA double-strand breaks (DSBs), which depend on the domesticated transposase PiggyMac. These DSBs all exhibit a characteristic geometry, with 4-base 5' overhangs centered on the conserved TA, and may readily align and undergo ligation with minimal processing.

The conjugation-specific Die5 protein is required for development of the somatic nucleus in both Paramecium and Tetrahymena.

Development in ciliated protozoa involves extensive genome reorganization within differentiating macronuclei, which shapes the somatic genome of the next vegetative generation. Major events of macronuclear differentiation include excision of internal eliminated sequences (IESs), chromosome fragmentation, and genome amplification. Proteins required for these events include those with homology throughout eukaryotes as well as proteins apparently unique to ciliates.

Coordinate regulation of a family of promastigote-enriched mRNAs by the 3'UTR PRE element in Leishmania mexicana.

Post-transcriptional regulation is a key feature controlling gene expression in the protozoan parasite Leishmania. The nine-nucleotide paraflagellar rod regulatory element (PRE) in the 3'UTR of Leishmania mexicana PFR2 is both necessary and sufficient for the observed 10-fold higher level of PFR2 mRNA in promastigotes compared to amastigotes. It is also found in the 3'UTRs of all known PFR genes.

Global gene expression in Leishmania.

The completion of the genomic sequences of many protozoan pathogens of humans, including species of Leishmania, Trypanosoma and Plasmodium, provide new approaches to study the pattern of gene expression during differentiation and development. Leishmania are a major public health risk in many countries and cause a wide spectrum of clinical disease referred to as leishmaniasis. The Leishmania life cycle consists of two morphologically distinct stages: intracellular amastigotes that reside in the phagolysosome of mammalian macrophages, and extracellular promastigotes that reside within the gut of the sandfly vector.

The SUMO pathway is developmentally regulated and required for programmed DNA elimination in Paramecium tetraurelia.

Extensive genome-wide remodeling occurs during the formation of the somatic macronuclei from the germ line micronuclei in ciliated protozoa. This process is limited to sexual reproduction and includes DNA amplification, chromosome fragmentation, and the elimination of internal segments of DNA. Our efforts to define the pathways regulating these events revealed a gene encoding a homologue of ubiquitin activating enzyme 2 (UBA2) that is upregulated at the onset of macronuclear development in Paramecium tetraurelia.

Expression profiling by whole-genome interspecies microarray hybridization reveals differential gene expression in procyclic promastigotes, lesion-derived amastigotes, and axenic amastigotes in Leishmania mexicana.

We examined the Leishmania mexicana transcriptome to identify differentially regulated mRNAs using high-density whole-genome oligonucleotide microarrays designed from the genome data of a closely related species, Leishmania major. Statistical analysis on array hybridization data representing 8156 predicted coding regions revealed 288 genes (3.5% of all genes) whose steady-state mRNA levels meet criteria for differential regulation between promastigotes and lesion-derived amastigotes.

Analysis of Paramecium tetraurelia A-51 surface antigen gene mutants reveals positive-feedback mechanisms for maintenance of expression and temperature-induced activation.

In Paramecium tetraurelia, variable surface antigen loci show mutually exclusive expression which is controlled primarily at the transcriptional level. Clonally stable expression of a single antigen has attracted models involving self-regulation by their gene products. However, direct demonstration of self-feedback at the molecular level has been complicated due to the inability to separate the functional gene from its product as well as copy number effects associated with injected extrachromosomal DNA in the polygenomic somatic nucleus.

The two cytoplasmic dynein-2 isoforms in Leishmania mexicana perform separate functions.

Eukaryotic organisms with cilia or flagella typically express two non-axonemal or "cytoplasmic" dyneins, dynein-1 and dynein-2. Interestingly, we find that Leishmania mexicana is unusual and contains two distinct cytoplasmic dynein-2 heavy chain genes (designated LmxDHC2.1 and LmxDHC2.

Identification of single nucleotide mutations that prevent developmentally programmed DNA elimination in Paramecium tetraurelia.

The excision of internal eliminated sequences (IESs) occurs during the differentiation of a new somatic macronuclear genome in ciliated protozoa. In Paramecium tetraurelia, IESs show few conserved features with the exception of an invariant 5'-TA-3' dinucleotide that is part of an 8-bp inverted terminal repeat consensus sequence with similarity to the ends of mariner/Tc1 transposons. We have isolated and analyzed two mutant cell lines that are defective in excision of individual IESs in the A-51 surface antigen gene.

Identification of a developmentally regulated translation elongation factor 2 in Tetrahymena thermophila.

Protein synthesis elongation factor 2 (eEF2) catalyzes the translocation of the peptidyl-tRNA from the A site to the P site of the ribosome. Most organisms encode a single EF2 protein and its activity is regulated by phosphorylation. We have identified a family of genes in Tetrahymena thermophila that encode proteins homologous to eEF2, yet are expressed only during sexual reproduction.