A Forward Genetic Screen Reveals that Calcium-dependent Protein Kinase 3 Regulates Egress in Toxoplasma

Egress from the host cell is a crucial and highly regulated step in the biology of the obligate intracellular parasite, Toxoplasma gondii. Active egress depends on calcium fluxes and appears to be a crucial step in escaping the attack from the immune system and, potentially, in enabling the parasites to shuttle into appropriate cells for entry into the brain of the host. Previous genetic screens have yielded mutants defective in both ionophore-induced egress and ionophore-induced death. Using whole genome sequencing of one mutant and subsequent analysis of all mutants from these screens, we find that, remarkably, four independent mutants harbor a mis-sense mutation in the same gene, TgCDPK3, encoding a calcium-dependent protein kinase. All four mutations are predicted to alter key regions of TgCDPK3 and this is confirmed by biochemical studies of recombinant forms of each. By complementation we confirm a crucial role for TgCDPK3 in the rapid induction of parasite egress and we establish that TgCDPK3 is critical for formation of latent stages in the brains of mice. Genetic knockout of TgCDPK3 confirms a crucial role for this kinase in parasite egress and a non-essential role for it in the lytic cycle.     

The phylogenetic potential of entire 26S rDNA sequences in plants

18S ribosomal RNA genes are the most widely used nuclear sequences for phylogeny reconstruction at higher taxonomic levels in plants. However, due to a conservative rate of evolution, 18S rDNA alone sometimes provides too few phylogenetically informative characters to resolve relationships adequately. Previous studies using partial sequences have suggested the potential of 26S or large-subunit (LSU) rDNA for phylogeny retrieval at taxonomic levels comparable to those investigated with 18S rDNA. Here we explore the patterns of molecular evolution of entire 26S rDNA sequences and their impact on phylogeny retrieval. We present a protocol for PCR amplification and sequencing of entire (approximately 3.4 kb) 26S rDNA sequences as single amplicons, as well as primers that can be used for amplification and sequencing. These primers proved useful in angiosperms and Gnetales and likely have broader applicability. With these protocols and primers, entire 26S rDNA sequences were generated for a diverse array of 15 seed plants, including basal eudicots, monocots, and higher eudicots, plus two representatives of Gnetales. Comparisons of sequence dissimilarity indicate that expansion segments (or divergence domains) evolve 6.4 to 10.2 times as fast as conserved core regions of 26S rDNA sequences in plants. Additional comparisons indicate that 26S rDNA evolves 1.6 to 2.2 times as fast as and provides 3.3 times as many phylogenetically informative characters as 18S rDNA; compared to the chloroplast gene rbcL, 26S rDNA evolves at 0.44 to 1.0 times its rate and provides 2.0 times as many phylogenetically informative characters. Expansion segment sequences analyzed here evolve 1.2 to 3.0 times faster than rbcL, providing 1.5 times the number of informative characters. Plant expansion segments have a pattern of evolution distinct from that found in animals, exhibiting less cryptic sequence simplicity, a lower frequency of insertion and deletion, and greater phylogenetic potential.      

Evidence for compartmentalized adenylate kinase catalysis serving a high energy phosphoryl transfer function in rat skeletal muscle

The first characterization of the kinetics and subcellular compartmentation of adenylate kinase activity in intact muscle has been accomplished using rat diaphragm equilibrated with [18O]water. Rates of adenylate kinase-catalyzed phosphoryl transfer were measured by appearance of 18O-labeled beta-phosphoryls in ADP and ATP resulting from the transfer to AMP of newly synthesized 18O-labeled gamma-ATP. Unique features of adenylate kinase catalysis were uncovered in the intact cell not predictable from cell free analysis. This enzyme activity, which in non-contracting muscle is limited to 1/1000 of the estimated Vmax (cell free) apparently because of restricted ADP availability, is localized in subcellular compartments that increase in size and/or number with contractile frequency. Contraction also causes frequency-dependent increments in adenylate kinase velocity (22-fold at 4 Hz) as does oxygen deprivation (35-fold). These enhanced rates of adenylate kinase activity, equivalent to processing all the cellular ATP and ADP in approximately 1 min, occur when levels of ATP, ADP, and AMP are maintained very near their basal steady state. These characteristics of the dynamics of adenylate kinase catalysis in the intact cell demonstrate that rapid rates of AMP production from ADP are balanced by equally rapid rates of AMP phosphorylation with no net synthesis or accumulation of any adenine nucleotide. This rapid processing of nucleotide phosphoryls conforms to a proposed scheme whereby the adenylate kinase system provides the unique function of transferring, as beta-ADP, high energy phosphoryls generated by glycolytic metabolism to ATP-utilizing components in muscle.     

Characterization of mutations and loss of heterozygosity of p53 and K-<Emphasis Type="Italic">ras</Emphasis>2 in pancreatic cancer cell lines by immobilized polymerase chain reaction

Background  

The identification of known mutations in a cell population is important for clinical applications and basic cancer research. In this work an immobilized form of the polymerase chain reaction, referred to as polony technology, was used to detect mutations as well as gene deletions, resulting in loss of heterozygosity (LOH), in cancer cell lines. Specifically, the mutational hotspots in p53, namely codons 175, 245, 248, 249, 273, and 282, and K-ras2, codons 12, 13 and 61, were genotyped in the pancreatic cell line, Panc-1. In addition LOH analysis was also performed for these same two genes in Panc-1 by quantifying the relative gene copy number of p53 and K-ras2.     

Ultra-high Pressure Decontamination of Insect Biocontrol Preparations

Living bacteria or bacterial spores are undesirable components of baculovirus preparations (e.g. granulosis virus) produced in vivo. This is also true, at least in Germany, for preparations of Bacillus thuringiensis subsp. tenebrionis. Using ultra-high pressure treatment (at a pressure of 550-580 MPa and a temperature of 50 C) the concentration of these bacterial contaminants was reduced by up to eight orders of magnitude. The biocontrol activity of both preparations remained unchanged.     

Mechanism of in vitro collagen fibril assembly. Kinetic and morphological studies

The kinetics of in vitro fibril assembly of Type I collagen preparations that contain different amounts of covalently cross-linked oligomers was studied with turbidimetry. Fibril formation showed a lag phase with no solution turbidity and a growth phase with a sigmoidal increase in the solution turbidity. The length of the lag phase was inversely related to both the total collagen concentration and the amount of covalently cross-linked oligomers in the solution. Double logarithmic plots of t1/4, the amount of time it takes for 1/4 of the collagen to assemble into fibrils, versus the total collagen concentration were linear but the slope decreased from -0.84 to -2.3 with decreasing amounts of covalently cross-linked oligomers in the samples. Electron microscopy showed the formation of unbanded microfibrils with diameters in the range of 3-15 nm early in the lag phase and larger diameter banded fibrils coexisting with the microfibrils near the end of the lag phase. Centrifugation of the solution at the lag phase prolonged the lag time, presumably by removal of microfibrils, but subsequent growth of the fibrils was unaffected. The results suggest a cooperative nucleation-growth mechanism for the in vitro assembly of collagen fibrils which is consistent with the results of an equilibrium study of the fibril assembly reaction we reported earlier (Na, G. C., Butz, L. J., Bailey, D. G., and Carroll, R. J. (1986) Biochemistry 25, 958-966).     

Synaptotagmin VII as a plasma membrane Ca(2+) sensor in exocytosis

Synaptotagmins I and II are Ca(2+) binding proteins of synaptic vesicles essential for fast Ca(2+)-triggered neurotransmitter release. However, central synapses and neuroendocrine cells lacking these synaptotagmins still exhibit Ca(2+)-evoked exocytosis. We now propose that synaptotagmin VII functions as a plasma membrane Ca(2+) sensor in synaptic exocytosis complementary to vesicular synaptotagmins. We show that alternatively spliced forms of synaptotagmin VII are expressed in a developmentally regulated pattern in brain and are concentrated in presynaptic active zones of central synapses. In neuroendocrine PC12 cells, the C(2)A and C(2)B domains of synaptotagmin VII are potent inhibitors of Ca(2+)-dependent exocytosis, but only when they bind Ca(2+). Our data suggest that in synaptic vesicle exocytosis, distinct synaptotagmins function as independent Ca(2+) sensors on the two fusion partners, the plasma membrane (synaptotagmin VII) versus synaptic vesicles (synaptotagmins I and II).     

Co-expression of molecular chaperones does not improve the heterologous expression of mammalian G-protein coupled receptor expression in yeast

The limitations to high-level expression of integral membrane proteins are not well understood. The human A(2)a adenosine receptor (A(2)a) and mouse Substance P receptor (SPR) were individually expressed in S. cerevisiae to identify potential cellular bottlenecks for G-protein coupled receptors. In the yeast system, A(2)a was not N-linked glycosylated but was functional and plasma membrane-localized. A(2)a also contained an intramolecular disulfide bond. Substance P receptor was also not N-linked glycosylated in yeast, but, unlike A(2)a, SPR was intracellularly retained, nonfunctional, and did not appear to contain an intramolecular disulfide bond. Since both receptors contain N-linked glycosylation and disulfide bonds in mammalian systems, machinery responsible for interacting with these modifications was investigated-specifically, the potential interactions between the nascent receptor and ER-resident proteins were explored. The chaperones calnexin and protein disulfide isomerase were co-overexpressed with the GPCRs to determine the effect on total and active yields of A(2)a and SPR, as well as on receptor trafficking. The effect of co-expressing the chaperone BiP on the total yields of A(2)a as well as intracellular fates of both receptors were determined. The co-expression of ER resident proteins did not improve A(2)a yields nor did they restore SPR activity or improve SPR cell surface expression. Taken together, these results indicate that an ER-folding bottleneck does not limit the expression of the mammalian receptors in yeast.     

Detecting changes in the relative expression of KRAS2 splice variants using polymerase colonies

Because splice variants of a gene with multiple isoforms give rise to proteins with different functions, it seems plausible that changes in the expression levels of the splice variants could be a contributing factor to disease. In fact, recent examples in the literature clearly illustrate that altered expression levels of splice variants may play an important role in disease. Furthermore, these works demonstrate that changes in expression levels could potentially be used to (1) monitor disease progression, (2) diagnose disease, and/or (3) determine disease state. In this work an immobilized form of PCR, known as polony technology, was adapted to quantify the relative expression levels of splice variants. Specifically, the relative expression levels of the two splice variants of the oncogene K-ras, namely, K-RAS2A and K-RAS2B, were determined using polony technology.