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The ongoing biotechnological revolution is rooted in our knowledge of enzymes. However, metagenomics is showing how little we know about Earth's enzyme repertoire. Deep sequencing has revolutionized our view of the tree of life. The genomes of newly‐discovered organisms are replete with novel sequences, emphasizing the trove of enzyme structures and functions waiting to be explored by biochemists. Here, we sought to draw attention to the vastness of the “enzymatic dark matter” within the tree of life by placing enzymological knowledge in the context of phylogeny. We used kinetic parameters from the BRaunschweig ENzyme DAtabase (BRENDA) as our proxy for enzymological knowledge. Mapping 12,677 BRENDA entries onto the phylogenetic tree revealed that 55% of these data were from eukaryotes, even though they are the least diverse part of the tree. At the next taxonomic level, only four of 18 archaeal phyla and 24 of 111 bacterial phyla are represented in the BRENDA dataset. One phylum, the Proteobacteria, accounts for over half of all bacterial entries. Similarly, the supergroup Amorphea, which includes animals and fungi, contains over half the data on eukaryotes. Many major taxonomic groups are notable for their complete absence from BRENDA, including the ultra‐diverse bacterial Candidate Phyla Radiation. At the species level, five mammals (including human) contribute 15% of BRENDA entries. The taxonomic bias in enzymology is strong, but in the era of gene synthesis we now have the tools to address it. Doing so promises to enrich our biochemical understanding of life and uncover powerful new biocatalysts.

A gene encoding creatine kinase was identified in two cryptosporidia species, Cryptosporidium muris and C. andersonii. They were syntenic and shared 91% identity 94% identity at the amino acid level and nucleotide levels respectively. The C. muris creatine kinase was characterized biochemically and shown to phosphorylate both creatine and glycocyamine with a 20-fold greater preference for creatine. The observed catalytic turnover with creatine was kcat = 30 s−1 with a catalytic efficiency of 15.4 mM−1 s−1. These values were within the range observed for other creatine kinases. A search of all the apicomplexa genomes available on EuPathDB did not reveal any other phosphagen kinase genes raising the possibility of horizontal gene transfer. However, no definitive conclusion could be drawn regarding this hypothesis given the massive amount of gene loss in the apicomplexa species which are primarily parasitic species. The implications of a creatine kinase in the parasites' infection cycle are discussed.

Phosphagen kinases (PKs) are known to be distributed throughout the animal kingdom, but have recently been discovered in some protozoan and bacterial species. A recent search of the available bacterial genomes revealed 49 unique sequences that appear to code for an arginine kinase (AK). The distribution of sequences was highly skewed with thirty nine out the forty nine sequences being found in six Proteobacteria classes (α, β, δ, γ, e, and ζ) which represented 46.6% of the 61,335 bacterial genomes available at JGI-IMG/M website. Moreover, twenty one of the unique and metagenome bAK sequences identified were from δ-Proteobacteria despite these representing only 0.88% of the total genomes available. Phylogenetic analyses revealed that the bacterial AK sequences were interpersed between basal species such as cnidarians, sponges and protozoa, displaying an unstable clustering that was dependent upon the parameters chosen for phylogenetic analysis. Three of these putative bacterial AK genes were cloned into the pET45 expression vector, expressed, and biochemically confirmed to be capable of phosphorylating arginine using ATP. Results of the kinetic analyses of the putative bAKs from Ahrensia, D. autotrophicum, and O. profundus show that the catalytic efficiencies with respect to arginine for each enzyme, measured at 104–105 M−1 s−1, fall within the range expected for competent arginine kinases.

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/KMArg) from (3.1 ± 0.6) × 104 to (9 ± 4) × 105 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.

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 × 105 M− 1 s− 1) and glycocyamine phosphorylation only weakly (kcat/KM (glycocyamine) = 2 × 102 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.

We discuss a pedagogical strategy to improve the writing of laboratory reports. Our multipronged approach gauges students' comprehension beforehand, rewards early success, and focuses feedback on the most serious writing issues. In preliminary studies, more students were able to achieve our threshold for success in our introductory courses, and students reported being more motivated to write well.

We characterized the calcineurin ( CaN ) gene family, including the subunits CaNA and CaNB , based upon sequence information obtained from the Paramecium genome project. Paramecium tetraurelia has seven subfamilies of the catalytic CaNA subunit and one subfamily of the regulatory CaNB subunit, with each subfamily having two members of considerable identity on the amino acid level (≥55% between subfamilies, ≥94% within CaNA subfamilies, and full identity in the CaNB subfamily). Within CaNA subfamily members, the catalytic domain and the CaNB binding region are highly conserved and molecular modeling revealed a three-dimensional structure almost identical to a human ortholog. At 14 members, the size of the CaNA family is unprecedented, and we hypothesized that the different CaNA subfamily members were not strictly redundant and that at least some fulfill different roles in the cell. This was tested by selecting two phylogenetically distinct members of this large family for posttranscriptional silencing by RNA interference. The two targets resulted in differing effects in exocytosis, calcium dynamics, and backward swimming behavior that supported our hypothesis that the large, highly conserved CaNA family members are not strictly redundant and that at least two members have evolved diverse but overlapping functions. In sum, the occurrence of CaN in Paramecium spp., although disputed in the past, has been established on a molecular level. Its role in exocytosis and ciliary beat regulation in a protozoan, as well as in more complex organisms, suggests that these roles for CaN were acquired early in the evolution of this protein family.

Phosphagen kinases (PKs) are a family of enzymes that catalyze a reversible phosphoryl transfer from ATP to various guanidino compounds. While PK substrate specificity is currently well understood,...

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.

A method utilizing electroporation to deliver antisense oligodeoxynucleotides into Paramecium tetraurelin has been developed. For these studies antisense oligonucleotides directed to different regions of the calrnodulin mRNA were used. It was found that a pulse delivered at 150-250 V ( 375-625 V/cm field strength) for 3.9-4.2 ms using a 275 pF capacitor with resistance set at 13 Ohms was sufficient to achieve measurable incorporation of fluorescently-labeled oligodeoxynucleotides in up to 95% of the cells treated. Optimal parameters included using oligodeoxynucleotides of at least 12 bases in length with a 3' blocking group at a dose of around 10 )LM. In addition, multiple oligodeoxynucleotides directed to the same target rnRNA resulted in at least a 10-fold reduction in the dose of oligodeoxynucleotide required to achieve the desired effects. Taken together, these results indicate that the use of antisense oligodeoxynucleotides can be an easy and useful method for linking genes to specific functions in Paramecium tetraurelia. Finally, this report discusses how different 3' blocking groups and the use of combinations of oligodeoxynucleotides directed to different regions of the same target mRNA can help address concerns about specificity. Supplementary key words. Calcium-dependent K' channel, calcium-dependent Na' channel, calmodulin, cholesterol-linked oligo- deoxynucleotides, hexanol-linked oligodeoxynucleotides, protozoan behavior. NTISENSE oligodeoxynucleotides (ODN) can be used to A transiently down-regulate a gene's expression and thereby help identify the role of the gene product in the cell (2). Their ease of use makes antisense ODN an attractive method for uti- lizing a "reverse genetics" approach for the assignment of function to cloned genes. Previously, we described the micro- injection of antisense ODN as a viable methodology in the cil- iated protozoan, Paramecium tetraurelia (5). In that report, an- tisense ODN were targeted against calmodulin mRNA resulting in abbreviated behavioral responses that mimicked the type of behavioral responses seen in cells with a mutant calmodulin gene that does not properly activate the calcium-dependent Na+ current. Thus, it was concluded that the antisense ODN reduced the levels of calmodulin sufficient to affect the activation of the calcium-dependent Na+ current. Recently it was reported that electroporation can be used as a method for incorporating ODN into cells (l) and, in an effort to improve the ease of use of this technology in ciliates, we have developed a procedure for introducing antisense ODN by electroporation. Calmodulin was again chosen as the target be- cause of its well-characterized role in the control of behavior in Paramecium (6, 141. In addition, we have characterized some of the parameters important for the use of this methodology in Paramecium.

An endogenous nuclear enzyme, RNase H, is an important component in determining the efficacy of antisense oligodeoxynucleotides (ODNs). In an effort to improve the potency of antisense ODNs, conjugates with three different nuclear targeting signal peptides were prepared. These short peptide sequences have been shown to facilitate transport of macromolecules into the nucleus of cells. Efficient chemistry for the synthesis of ODN-peptide conjugates is described. Reaction of 5'-aminohexyl-modified ODNs with iodoacetic anhydride gave pure iodoacetamide ODNs (IA-ODNs) in good yield. These electrophilic intermediates were reacted with thiol-containing peptides to give ODN-peptides in excellent yield and purity. The ODN-peptides were further characterized by proteolysis with trypsin. Thermal denaturation studies with ssDNA targets showed little effect of the 5'-peptide modifications on the hybridization properties of the ODN. The effect of the nuclear signal peptides on antisense potency was evaluated in the freshwater ciliate Paramecium. A 3'-hexanol-modified 24-mer antisense ODN, complementary to the mRNA for calmodulin, alters regulation of membrane ion channels and swimming behavior of these cells. A 2'-O-methyl analog of this ODN was inactive, thus providing evidence that this activity in Paramecium is mediated by RNase H. Antisense ODN-nuclear signal peptide conjugates were transfected into the cells by electroporation. Surprisingly, these conjugates showed no antisense effects in comparison to a 5'-unmodified control ODN. Random peptides or amino acids conjugated to the 5'-terminus did not decrease antisense activity.

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.

Paramecium tetraurelia has the shortest known introns as its standard intron length. Sequenced introns vary between 20 and 33 nucleotides in length. The intron sequences were discovered in genomic sequences coding for a variety of different proteins, including phosphatases, kinases, and low-molecular weight GTP-binding proteins. All intron sequences begin with the conserved dinucleotide GT and end with the conserved dinucleotide AG. The sequences are more AT rich than the Paramecium coding sequences. The identified sequences were confirmed as introns by sequencing several cDNA fragments. We report here analysis of the characteristics of 50 separate introns, including size, base composition, and a consensus sequence.

The calcium-binding protein calmodulin has been shown to modulate the Ca(2+)-dependent ion channels of Paramecium tetraurelia. Mutations in the calmodulin gene of Paramecium result in an altered pattern of behavioral responses. Antisense oligodeoxyribonucleotides (ODNs), complementary to calmodulin mRNA in Paramecium, were synthesized from a modified solid support that introduced a 3'-hydroxyhexyl phosphate. These 3'-modified ODNs were tested for their ability to alter the behavioral response of Paramecium. The microinjection of antisense ODNs temporarily reduced the backward swimming behavior of the cells in test solutions containing Na+. The injection of sense and random 3'-modified ODNs, or unmodified antisense ODNs, had no effect. The antisense ODN-induced effect was reversed by the injection of calmodulin protein. The pattern of response of the injected cells in various behavioral test solutions indicated that the calmodulin antisense ODNs reduce the Ca(2+)-dependent Na+ current. Antisense ODNs, complementary either to the 5' start site or to an internal sequence of the calmodulin mRNA, were similarly effective in altering behavior. These results show that antisense ODNs may be utilized in ciliated protozoa as a tool for reducing the expression of specific gene products. In addition, Paramecium represents a powerful model system with which to study and develop antisense ODN technology.

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.

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. Curr. Protoc. Essential Lab. Tech. 8:10.3.1-10.3.40. © 2014 by John Wiley & Sons, Inc. Keywords: quantifying gene expression; real-time PCR (polymerase chain reaction); cDNA; PCR primer design; Taq polymerase; SYBR Green; PCR

Eight variants of creatine kinase were created to switch the substrate specificity from creatine to glycocyamine using a rational design approach. Changes to creatine kinase involved altering several residues on the flexible loops that fold over the bound substrates including a chimeric replacement of the guanidino specificity loop from glycocyamine kinase into creatine kinase. A maximal 2,000-fold change in substrate specificity was obtained as measured by a ratio of enzymatic efficiency (k(cat)/K(M).K(d)) for creatine vs. glycocyamine. In all cases, a change in specificity was accompanied by a large drop in enzymatic efficiency. This data, combined with evidence from other studies, indicate that substrate specificity in the phosphagen kinase family is obtained by precise alignment of substrates in the active site to maximize k(cat)/K(M).K(d) as opposed to selective molecular recognition of one guanidino substrate over another. A model for the evolution of the dimeric forms of phosphagen kinases is proposed in which these enzymes radiated from a common ancestor that may have possessed a level of catalytic promiscuity. As mutational events occurred leading to greater degrees of substrate specificity, the dimeric phosphagen kinases became evolutionary separated such that the substrate specificity could not be interchanged by a small number of mutations.

Arginine kinases (AK) are members of the phosphagen kinase family and like other phosphagen kinases, they help in regulating energy metabolism in the cell. They catalyze the transfer of a high-ener...

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.

We have used the polymerase chain reaction to clone a large number of gene fragments encoding low-molecularweight GTP-binding proteins (LMW G-proteins) from Paramecium tetraurelia . All clones were subjected to a computerassisted search of the GenBank databases to assign putative homologues. On the basis of several conserved features, we place these genes in the rab/ypt and rho subfamilies of the LMW G-proteins. Northern blot analyses indicated that all clones were from active genes. One surprising feature of the cloned genomic sequences was the presence of unusually short introns (20–30 nucleotides).