We have isolated three alleles of a novel Drosophila clock gene, double-time (dbt). Short- (dbtS) and long-period (dbtL) mutants alter both behavioral rhythmicity and molecular oscillations from previously identified clock genes, period and timeless. A third allele, dbtP, causes pupal lethality and eliminates circadian cycling of per and tim gene products in larvae. In dbtP mutants, PER proteins constitutively accumulate, remain hypophosphorylated, and no longer depend on TIM proteins for their accumulation. We propose that the normal function of DOUBLETIME protein is to reduce the stability and thus the level of accumulation of monomeric PER proteins. This would promote a delay between per/tim transcription and PER/TIM complex function, which is essential for molecular rhythmicity.

Circadian (24 hour) PERIOD (PER) protein oscillation is dependent on the double-time (dbt) gene, a casein kinase Ivarepsilon homolog [1-3]. Without dbt activity, hypophosphorylated PER proteins over-accumulate, indicating that dbt is required for PER phosphorylation and turnover [3,4]. There is evidence of a similar role for casein kinase Ivarepsilon in the mammalian circadian clock [5,6]. We have isolated a new dbt allele, dbt(ar), which causes arrhythmic locomotor activity in homozygous viable adults, as well as molecular arrhythmicity, with constitutively high levels of PER proteins, and low levels of TIMELESS (TIM) proteins. Short-period mutations of per, but not of tim, restore rhythmicity to dbt(ar) flies. This suppression is accompanied by a restoration of PER protein oscillations. Our results suggest that short-period per mutations, and mutations of dbt, affect the same molecular step that controls nuclear PER turnover. We conclude that, in wild-type flies, the previously defined PER'short domain' [7,8] may regulate the activity of DBT on PER.

The dynamic evolution of organelle compartmentalization in eukaryotes and how strictly compartmentalization is maintained are matters of ongoing debate. While the endoplasmic reticulum (ER) is classically envisioned as the site of protein cotranslational translocation, it has recently been proposed to have pluripotent functions. Using transfected reporter constructs, organelle-specific markers, and functional enzyme assays, we now show that in an early-diverging protozoan, Giardia lamblia, endocytosis and subsequent degradation of exogenous proteins occur in the ER or in an adjacent and communicating compartment. The Giardia endomembrane system is simple compared to those of typical eukaryotes. It lacks peroxisomes, a classical Golgi apparatus, and canonical lysosomes. Giardia orthologues of mammalian lysosomal proteases function within an ER-like tubulovesicular compartment, which itself can dynamically communicate with clathrin-containing vacuoles at the periphery of the cell to receive endocytosed proteins. These primitive characteristics support Giardia's proposed early branching and could serve as a model to study the compartmentalization of endocytic and lysosomal functions into organelles distinct from the ER. This system also may have functional similarity to the retrograde transport of toxins and major histocompatibility complex class I function in the ER of mammals.

Giardia lamblia is a protozoan parasite and the earliest branching clade of eukaryota. The Giardia life cycle alternates between an asexually replicating vegetative form and an infectious cyst form. Encystation and excystation are crucial processes for the survival and transmission of Giardia. Cysteine proteases in Giardia have been implicated in proteolytic processing events that enable the continuance of the life cycle throughout encystation and excystation. Using quantitative real-time PCR, the expression of twenty-seven clan CA cysteine protease genes in the Giardia genome was measured during both vegetative growth and encystation. Giardia cysteine protease 2 was the most highly expressed cysteine protease during both life cycle stages measured, with a dramatic expression increase during encystation. The mRNA transcript for Giardia cysteine protease 2 was 7-fold up-regulated during encystation and was greater than 3-fold higher than any other Giardia protease gene product. Recombinant Giardia cysteine protease 2 was expressed, purified, and biochemically characterized. The activity of the recombinant cysteine protease 2 protein was confirmed to be identical to the dominant cysteine protease activity found in G. lamblia lysates. Giardia cysteine protease 2 was co-localized with cyst wall protein in encystation-specific vesicles during encystation and processed cyst wall protein 2 to the size found in Giardia cyst walls. These data suggest that Giardia cysteine protease 2 is not only the major cysteine endoprotease expressed in Giardia, but is also central to the encystation process.Giardia lamblia is a protozoan parasite that inhabits the upper small intestine of many vertebrate hosts and is the most commonly isolated intestinal parasite world wide (1). In addition to its medical importance, Giardia is of interest as a model cell system because it represents the most early branching clade of eukaryota (2, 3). Giardia has a simple two-stage life cycle consisting of a vegetative replicating trophozoite and an infectious cyst. Infection is initiated with cyst ingestion by a vertebrate host. After passage through the acidic host stomach, vegetative trophozoites emerge from the cyst by the process of excystation, asexually divide by binary fission, establish the duodenal infection, and give rise to the characteristic symptoms of giardiasis. Trophozoites can form infective cysts that are passed in the host feces and ingested by another host to propagate the life cycle (1).The process of encystation is a coordinated secretion of cyst wall materials to the periphery of a cell to form the cyst wall (4, 5). In response to environmental cues, trophozoites produce abundant cyst wall proteins that are packaged into encystationspecific vesicles (ESVs). 3 These vesicles grow, mature, and eventually traffic to the plasma membrane of the trophozoite, where cyst wall precursor material is secreted to form the environmentally stable cyst wall (4, 6, 7). The expression of many proteins is up-regulated during the encystation proc...

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