Dean Fraga

Real‐Time PCR

Real‐time PCR is a recent modification to the polymerase chain reaction that allows precise quantification of specific nucleic acids in a complex mixture by fluorescent detection of labeled PCR products. Detection can be accomplished using specific, as well as nonspecific fluorescent probes. Real‐time PCR is often used in the quantification of gene expression levels. Prior to using real‐time PCR to quantify a target message, care must be taken to optimize the RNA isolation, primer design, and PCR reaction conditions so that accurate and reliable measurements can be made. This short overview of real‐time PCR discusses basic principles behind real‐time PCR, some optimization and experimental design considerations, and how to quantify the data generated using both relative and absolute quantification approaches. Useful Web sites and texts that expand upon topics discussed are also listed.

Apoptotic cascades as possible targets for inhibiting cell death in Huntington’s disease

Huntington’s disease (HD) is a devastating autosomal dominant disorder characterized by progressive motor and neuropsychological symptoms. Evidence implicating the apoptotic cascades as a possible cause for the neurodegeneration seen in HD has directed researchers toward investigating therapeutic treatments targeting caspases and other proapoptotic factors. Cellular and murine models, which have demonstrated that caspase-mediated cleavage could be the cause for the neurodegeneration seen in HD, have evoked more research investigating the possible inhibition of apoptosis in HD. In particular, minocycline, a tetracycline-derived antibiotic that has been shown to increase survival in transgenic mouse models of HD, exhibits a neuroprotective feature in HD and demonstrates an anti-inflammatory as well as an anti-microbial effect by inhibiting microglial activation known to cause apoptosis.

Characterization of a novel bacterial arginine kinase from Desulfotalea psychrophila

Phosphagen kinases are found throughout the animal kingdom and catalyze the transfer of a high-energy gamma phosphoryl-group from ATP to a guanidino group on a suitable acceptor molecule such as creatine or arginine. Recent genome sequencing efforts in several proteobacteria, including Desulfotalea psychrophila LSv54, Myxococcus xanthus, Sulfurovum sp. NBC37-1, and Moritella sp. PE36 have revealed what appears to be a phosphagen kinase homolog present in their genomes. Based on sequence comparisons these putative homologs bear a strong resemblance to arginine kinases found in many invertebrates and some protozoa. We describe here a biochemical characterization of one of these homologs from D. psychrophila expressed in E. coli that confirms its ability to reversibly catalyze phosphoryl transfer from ATP to arginine. A phylogenetic analysis suggests that these bacteria homologs are not widely distributed in proteobacteria species. They appear more related to protozoan arginine kinases than to similar proteins seen in some Gram-positive bacteria that share key catalytic residues but encode protein tyrosine kinases. This raises the possibility of horizontal gene transfer as a likely origin of the bacterial arginine kinases.

Identification and Characterization of a Putative Arginine Kinase Homolog from Myxococcus xanthus Required for Fruiting Body Formation and Cell Differentiation

Arginine kinases catalyze the reversible transfer of a high-energy phosphoryl group from ATP to l-arginine to form phosphoarginine, which is used as an energy buffer in insects, crustaceans, and some unicellular organisms. It plays an analogous role to that of phosphocreatine in vertebrates. Recently, putative arginine kinases were identified in several bacterial species, including the social Gram-negative soil bacterium Myxococcus xanthus. It is still unclear what role these proteins play in bacteria and whether they have evolved to acquire novel functions in the species in which they are found. In this study, we biochemically purified and characterized a putative M. xanthus arginine kinase, Ark, and demonstrated that it has retained the ability to catalyze the phosphorylation of arginine by using ATP. We also constructed a null mutation in the ark gene and demonstrated its role in both certain stress responses and development.

Characterization of a putative oomycete taurocyamine kinase: Implications for the evolution of the phosphagen kinase family

Phosphagen kinases (PKs) are known to be distributed throughout the animal kingdom, but have recently been discovered in some protozoan and bacterial species. Within animal species, these enzymes play a critical role in energy homeostasis by catalyzing the reversible transfer of a high-energy phosphoryl group from Mg⋅ATP to an acceptor molecule containing a guanidinium group. In this work, a putative PK gene was identified in the oomycete Phytophthora sojae that was predicted, based on sequence homology, to encode a multimeric hypotaurocyamine kinase. The recombinant P. sojae enzyme was purified and shown to catalyze taurocyamine phosphorylation efficiently (kcat/KM (taurocyamine) = 2 × 10(5) M(-1) s(-1)) and glycocyamine phosphorylation only weakly (kcat/KM (glycocyamine) = 2 × 10(2) M(-1) s(-1)), but lacked any observable kinase activity with the more ubiquitous guanidinium substrates, creatine or arginine. Additionally, the enzyme was observed to be dimeric but lacked cooperativity between the subunits in forming a transition state analog complex. These results suggest that protozoan PKs may exhibit more diversity in substrate specificity than was previously thought.

Characterization of the arginine kinase isoforms in Caenorhabditis elegans.

Phosphagen kinases (PKs) are well-studied enzymes involved in energy homeostasis in a wide range of animal, protozoan, and even some bacterial species. Recent genome efforts have allowed comparative work on the PKs to extend beyond the biochemistry of individual proteins to the comparative cellular physiology and examining of the role of all PK family members in an organism. The sequencing of the Caenorhabditis elegans genome and availability of sophisticated genetic tools within that system affords the opportunity to conduct a detailed physiological analysis of the PKs from a well known invertebrate for comparison with the extensive work conducted on vertebrate systems. As a first step in this effort we have carried out a detailed molecular genetic and biochemical characterization of the PKs in C. elegans. Our results reveal that C. elegans has five PK genes encoding arginine kinases that range in catalytic efficiency (kcat/KM(Arg)) from (3.1±0.6)×10(4) to (9±4)×10(5) M(-1) s(-1). This range is generally within the range seen for arginine kinases from a variety of species. Our molecular genetic and phylogenetic analysis reveals that the gene family has undergone extensive intron loss and gain within the suborder Rhabditina. In addition, within C. elegans we find evidence of gene duplication and loss. The analysis described here for the C. elegans AKs represents one of the most complete biochemical and molecular genetic analysis of a PK family within a genetically tractable invertebrate system and opens up the possibility of conducting detailed physiological comparisons with vertebrate systems using the sophisticated tools available with this model invertebrate system.

The Particle Inflow Gun can be used to Co‐transform Paramecium using Tungsten Particles

ABSTRACT. A particle inflow gun (PIG) was constructed and tested for its utility to transform Paramecium using tungsten or gold as the DNA carrier particle. In the first set of experiments we transformed Paramecium with a plasmid containing the neomycin‐resistance gene, obtaining a transformation efficiency of 0.31±0.14% (mean±SD) for tungsten particles and 1.30±0.29% for gold particles. Plasmid DNA precipitated upon tungsten was shown to be stable for transformation purposes for up to 1 h prior to use and had no detectable effects on transformation efficiency. In addition, we demonstrated that at high frequency (71±20%) a Paramecium mutant strain could be phenotypically rescued by co‐transformation with a second plasmid containing the selectable neomycin‐resistance gene. The PIG coupled with tungsten particles as the carrier offers a low‐cost alternative for biolistic transformation of Paramecium.