Isolation and characterization of a human cDNA for mRNA 5'-capping enzyme.

The human mRNA 5'-capping enzyme cDNA was identified. Three highly related cDNAs, HCE1 (human mRNAcappingenzyme1), HCE1A and HCE1B , were isolated from a HeLa cDNA library. The HCE1 cDNA has the longest ORF, which can encode a 69 kDa protein. A short region of 69 bp in the 3'-half of the HCE1 ORF was missing in HCE1A and HCE1B , and, additionally, HCE1B has an early translation termination signal, which suggests that the latter two cDNAs represent alternatively spliced product. When expressed in Escherichia coli as a fusion protein with glutathione S -transferase, Hce1p displayed both mRNA 5'-triphosphatase (TPase) and mRNA 5'-guanylyltransferase (GTase) activities, and it formed a cap structure at the 5'-triphosphate end of RNA, demonstrating that it indeed specifies an active mRNA 5'-capping enzyme. The recombinant proteins derived from HCE1A and HCE1B possessed only TPase activity. When expressed from ADH1 promoter, HCE1 but not HCE1A and HCE1B complemented Saccharomyces cerevisiae CEG1 and CET1 , the genes for GTase and TPase, respectively. These results demonstrate that the N-terminal part of Hce1p is responsible for TPase activity and the C-terminal part is essential for GTase activity. In addition, the human TPase domain cannot functionally substitute for the yeast enzyme in vivo.     

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

Males and hermaphrodites are the two naturally found sexual forms in the nematode C. elegans. The amoeboid sperm are produced by both males and hermaphrodites. In the earlier phase of gametogenesis, the germ cells of hermaphrodites differentiate into limited number of sperm - around 300 - and are stored in a small ''bag'' called the spermatheca. Later on, hermaphrodites continually produce oocytes¹. In contrast, males produce exclusively sperm throughout their adulthood. The males produce so much sperm that it accounts for >50% of the total cells in a typical adult worm². Therefore, isolating sperm from males is easier than from that of hermaphrodites.Only a small proportion of males are naturally generated due to spontaneous non-disjunction of X chromosome³. Crossing hermaphrodites with males or more conveniently, the introduction of mutations to give rise to Him (High Incidence of Males) phenotype are some of strategies through which one can enrich the male population³.Males can be easily distinguished from hermaphrodites by observing the tail morphology⁴. Hermaphrodite''s tail is pointed, whereas male tail is rounded with mating structures.Cutting the tail releases vast number of spermatids stored inside the male reproductive tract. Dissection is performed under a stereo microscope using 27 gauge needles. Since spermatids are not physically connected with any other cells, hydraulic pressure expels internal contents of male body, including spermatids².Males are directly dissected on a small drop of ''Sperm Medium''. Spermatids are sensitive to alteration in the pH. Hence, HEPES, a compound with good buffering capacity is used in sperm media. Glucose and other salts present in sperm media help maintain osmotic pressure to maintain the integrity of sperm.Post-meiotic differentiation of spermatids into spermatozoa is termed spermiogenesis or sperm activation. Shakes⁵, and Nelson⁶ previously showed that round spermatids can be induced to differentiate into spermatozoa by adding various activating compounds including Pronase E. Here we demonstrate in vitro spermiogenesis of C. elegans spermatids using Pronase E.Successful spermiogenesis is pre-requisite for fertility and hence the mutants defective in spermiogenesis are sterile. Hitherto several mutants have been shown to be defective specifically in spermiogenesis process⁷. Abnormality found during in vitro activation of novel Spe (Spermatogenesis defective) mutants would help us discover additional players participating in this event.Download video file.^{(33M, flv)}     

Cloning and characterization of antioxidant enzyme methionine sulfoxide-S-reductase from Caenorhabditis elegans

Methionine (Met) residues in proteins are susceptible to oxidation. The resulting methionine sulfoxide can be reduced back to methionine by methionine sulfoxide-S-reductase (MsrA). The MsrA gene, isolated from Caenorhabditis elegans, was cloned and expressed in Escherichia coli. The resultant enzyme was able to revert both free Met and Met in proteins in the presence of either NADPH or dithiothreitol (DTT). However, approximately seven times higher enzyme activity was observed in the presence of DTT than of NADPH. The enzyme had an absolute specificity for the reduction of l-methionine-S-sulfoxide but no specificity for the R isomer. K(m) and k(cat) values for the enzyme were approximately 1.18 mM and 3.64 min(-1), respectively. Other kinetics properties of the enzyme were also evaluated.     

In vitro interactions of Caenorhabditis elegans dystrophin with dystrobrevin and syntrophin

HeT-A elements are non-long terminal repeat retrotransposons added onto the Drosophila chromosome ends. We have investigated the formation in vitro of higher order structures by oligonucleotides derived from the 3' non-coding region of HeT-A elements and found that they are capable of forming G-quadruplex DNA. These results suggest that the 3' repeat region of HeT-A may structurally behave as the telomeric repeats common to a majority of eukaryotes. The presence of structural motifs shared by telomeres and centromeres and the implications of these findings for chromosome evolution are discussed.     

Lipase-like 5 enzyme controls mitochondrial activity in response to starvation in Caenorhabditis elegans

The C. elegans lipase-like 5 (lipl-5) gene is predicted to code for a lipase homologous to the human gastric acid lipase. Its expression was previously shown to be modulated by nutritional or immune cues, but nothing is known about its impact on the lipid landscape and ensuing functional consequences. In the present work, we used mutants lacking LIPL-5 protein and found that lipl-5 is important for normal lipidome composition as well as its remodeling in response to food deprivation. Particularly, lipids with signaling functions such as ceramides and mitochondrial lipids were affected by lipl-5 silencing. In comparison with wild type worms, animals lacking LIPL-5 were enriched in cardiolipins linked to polyunsaturated C20 fatty acids and coenzyme Q-9. Differences in mitochondrial lipid composition were accompanied by differences in mitochondrial activity as mitochondria from well-fed lipl-5 mutants were significantly more able to oxidize respiratory substrates when compared with mitochondria from well-fed wild type worms. Strikingly, starvation elicited important changes in mitochondrial activity in wild type worms, but not in lipl-5 worms. This indicates that this lipase is a determinant of mitochondrial functional remodeling in response to food withdrawal.     

mRNA surveillance mitigates genetic dominance in Caenorhabditis elegans

Nonsense mutant mRNAs are unstable in all eucaryotes tested, a phenomenon termed nonsense-mediated mRNA decay (NMD) or mRNA surveillance. Functions of the seven smg genes are required for mRNA surveillance in Caenorhabditis elegans. In Smg(+) genetic backgrounds, nonsense-mutant mRNAs are unstable, while in Smg(?) backgrounds such mRNAs are stable. Previous work has demonstrated that the elevated level of nonsense-mutant mRNAs in Smg(?) animals can influence the phenotypic effects of heterozygous nonsense mutations. Certain nonsense alleles of a muscle myosin heavy chain gene are recessive in Smg(+) backgrounds but strongly dominant in Smg(?) backgrounds. Such alleles probably express disruptive myosin polypeptide fragments whose abundance is elevated in smg mutants due to elevation of mRNA levels. We report here that mutations in a variety of C. elegans genes are strongly dominant in Smg(?), but recessive or only weakly dominant in Smg(+) backgrounds. We isolated 32 dominant visible mutations in a Smg(?) genetic background and tested whether their dominance requires a functional NMD system. The dominance of 21 of these mutations is influenced by NMD. We demonstrate, furthermore, that in the case of myosin, the dominant-negative effects of nonsense alleles are likely to be due to expression of N-terminal nonsense-fragment polypeptides, not to mistranslation of the nonsense codons. mRNA surveillance, therefore, may mitigate potentially deleterious effects of many heterozygous germline and somatic nonsense or frameshift mutations. We also provide evidence that smg-6, a gene previously identified as being required for NMD, performs essential function(s) in addition to its role in NMD.     

In vivo imaging of retrogradely transported synaptic vesicle proteins in Caenorhabditis elegans neurons

Axonal transport is an essential process that carries cargoes in the anterograde direction to the synapse and in the retrograde direction back to the cell body. We have developed a novel in vivo method to exclusively mark and dynamically track retrogradely moving compartments carrying specific endogenous synaptic vesicle proteins in the Caenorhabditis elegans model. Our method is based on the uptake of a fluorescently labeled anti-green fluorescent protein (GFP) antibody delivered in an animal expressing the synaptic vesicle protein synaptobrevin-1::GFP in neurons. We show that this method largely labels retrogradely moving compartments. Very little labeling is observed upon blocking vesicle exocytosis or if the synapse is physically separated from the cell body. The extent of labeling is also dependent on the dyenin-dynactin complex. These data support the interpretation that the labeling of synaptobrevin-1::GFP largely occurs after vesicle fusion and the major labeling likely takes place at the synapse. Further, we observe that the retrograde compartment carrying synaptobrevin contains synaptotagmin but lacks the endosomal marker RAB-5. This labeling method is very general and can be readily adapted to any transmembrane protein on synaptic vesicles with a GFP tag inside the vesicle and can also be extended to other model systems.     

Purification and characterization of beta-galactoside-binding proteins from Caenorhabditis elegans.

Two carbohydrate-binding proteins (subunit molecular masses, 32 and 16 kDa, respectively) were isolated for the first time from a nematode, Caenorhabditis elegans. They were specifically extracted with lactose and adsorbed on asialofetuin-Sepharose in the absence of a metal ion. Although these two proteins were co-eluted from a gel filtration column at a position corresponding to an apparent molecular size of 30 kDa under non-denaturing conditions, they could be separated by reversed-phase chromatography. The 32 kDa protein, the main component, was further characterized. Together with its solubility, saccharide specificity and metal independence, some other structural properties, including its amino acid composition, UV spectrum, and partial amino acid sequence, strongly suggested that the 32 kDa protein is a member of a class of soluble beta-galactoside-binding lectins which had previously been only found in vertebrates.     

In vitro and in vivo enzyme studies of polyhemoglobin-tyrosinase

Melanoma is now the fifth most common type of cancer in North America. At present, there is no optimal treatment for this cancer. However, the lowering of the tyrosine level can inhibit the growth of melanoma. Unfortunately, this diet restriction cannot be humanly tolerated and causes vomiting, nausea, and severe body weight loss. To prevent these problems, we are studying a new approach involving the preparation intermolecularly crosslinked hemoglobin and tyrosinase for intravenous injection. In this article we describe the method of preparation and the structural and functional properties of polyhemoglobin-tyrosinase. We evaluate the effects of varying glutaraldehyde ratio, crosslinking time, and enzyme concentration on the enzyme activity of polyhemoglobin-tyrosinase. We also optimize the molecular weight distribution of polyhemoglobin-tyrosinase. The stability of polyhemoglobin-tyrosinase at 37 degrees C is much more stable when compared to noncrosslinked tyrosinase solution. Animal studies show that a higher degree of polymerization correlates with a longer circulation time of polyhemoglobin-tyrosinase, and the optimal crosslinking time is 24 hours. One intravenous injection of polyhemoglobin-tyrosinase lowers the plasma tyrosine to about 10% of its original level within one hour.     

The pharynx of Caenorhabditis elegans.

The anatomy of the pharynx of Caenorhabditis elegans has been reconstructed from electron micrographs of serial sections. The pharynx is used for pumping food into the gut, and is composed of 34 muscle cells, 9 marginal cells, 9 epithelial cells, 5 gland cells and 20 neurones. Three regions of specialization in the cuticle lining of the pharyngeal lumen may aid in the accumulation of food particles. A basement membrane isolates the pharynx from the rest of the animal, making the pharyngeal nervous system a nearly self-contained unit which is composed primarily of five classes of motor neurones and six classes of interneurones. Three other classes have also been described, which by their morphology appear to be neurosecretory and motor, motor and interneuronal, and lastly one pair that only innervates three of the marginal cells. Some classes of neurone have free endings just under the cuticle lining the lumen of the pharynx, suggesting that these are mechano- or proprio-receptive endings. The connectivity of these neurones has been described at the level of individual synaptic regions, and after combining this information with video taped observations of the pharynx pumping, some interpretations of how these neurones function have been offered.     

Inhibition of mRNA translation extends lifespan in Caenorhabditis elegans

Protein synthesis is a regulated cellular process that links nutrients in the environment to organismal growth and development. Here we examine the role of genes that regulate mRNA translation in determining growth, reproduction, stress resistance and lifespan. Translational control of protein synthesis by regulators such as the cap-binding complex and S6 kinase play an important role during growth. We observe that inhibition of various genes in the translation initiation complex including ifg-1, the worm homologue of eIF4G, which is a scaffold protein in the cap-binding complex; and rsks-1, the worm homologue of S6 kinase, results in lifespan extension in Caenorhabditis elegans. Inhibition of ifg-1 or rsks-1 also slows development, reduces fecundity and increases resistance to starvation. A reduction in ifg-1 expression in dauers was also observed, suggesting an inhibition of protein translation during the dauer state. Thus, mRNA translation exerts pleiotropic effects on growth, reproduction, stress resistance and lifespan in C. elegans.     

Further characterization of a temperature-sensitive transformation mutant in Caenorhabditis elegans.

The temperature-sensitive sex transformer tra-2 (b202) II of the nematode Caenorhabditis elegans causes the transformation of genotypically hermaphrodite worms into phenotypic males and sterile intersexes at restrictive temperature. In this note, we show that the entire gonad structure is transformed and that oocyte development is autonomous of the form of the gonad and of the presence of a cellular sheath. Four oocyte-specific proteins are present in male intersexes that produce oocytes but are lacking in transformed males and hermaphrodite intersexes that do not produce oocytes.     

Purification and characterization of recombinant Caenorhabditis elegans metallothionein

The roundworm Caenorhabditis elegans adapted for survival at high concentrations of Cd(II) expresses two isoforms of metallothionein, CeMT-I and CeMT-II. To characterize one of these proteins CeMT-II was prepared as its Cd containing form by expressing its cDNA heterologously in Escherichia coli. The purified 63-amino-acid protein was identified as the desired product by ion-spray mass spectrometry and was found to resemble in most of its chemical and spectroscopic features the metallothioneins of other animal phyla. The recombinant protein contains a total of 18 cysteine residues and, as documented by electrophoresis and mass spectrometry, binds firmly six Cd ions through the cysteine's side chains. The (113)Cd NMR spectrum features six (113)Cd resonances. Their chemical shift positions between 615 and 675 ppm denote the existence of clusters of tetrahedrally coordinated cadmium thiolate complexes. The metal thiolate coordination dominates also the electronic far-UV absorption spectrum. It is characterized by a massive absorption profile with Cd thiolate shoulders at 255 and 235 nm. Upon replacement of Cd by Zn the profile was blue-shifted by 30 nm.     

Molecular characterization of the Caenorhabditis elegans Rho GDP-dissociation inhibitor.

GDP-dissociation inhibitors (GDIs) form one of the classes of regulatory proteins that modulate the cycling of the Ras superfamily of GTPases between active GTP-bound and inactive GDP-bound states. We report here the characterization of the Caenorhabditis elegans RhoGDI (CeRhoGDI) as part of our investigations into Rho-GTPase signalling pathways that are involved in nematode development. CeRhoGDI is a 23-kDa protein that is localized predominantly in the cytosol. CeRhoGDI interacts only with the lipid-modified forms of C. elegans Rho-GTPases, CeRhoA, CeRac1 and Cdc42Ce, in vitro and is able to solubilize the membrane-bound forms of these GTPases. CeRhoGDI recognizes the GTPases in both GTP- and GDP-bound forms; hence it inhibits both the guanine-nucleotide dissociation and GTP-hydrolysis activities. The inhibitory activity towards the GTP-bound GTPases is weak compared with that towards GDP-bound GTPases. CeRhoGDI is expressed throughout development and is highly expressed in marginal and vulval epithelial cells, in sperm cells and spicules. Taken together, our results suggest that CeRhoGDI may be involved in specific morphogenetic events mediated by the C. elegans Rho-GTPases.     

The astacin protein family in Caenorhabditis elegans.

In the nematode Caenorhabditis elegans, 40 genes code for astacin-like proteins (nematode astacins, NAS). The astacins are metalloproteases present in bacteria, invertebrates and vertebrates and serve a variety of physiological functions like digestion, hatching, peptide processing, morphogenesis and pattern formation. With the exception of one distorted pseudogene, all the other C. elegans astacins are expressed and are evidently functional. For 13 genes we found splicing patterns differing from the Genefinder predictions in WormBase, sometimes markedly. The GFP expression pattern for NAS-4 shows a specific localization in anterior pharynx cells and in the whole digestive tract (as the secreted form). In contrast, NAS-7 is found in the head of adult hermaphrodites, but not in pharynx cells or in the lumen of the digestive tract. In embryos, NAS-7 fluorescence becomes detectable just before hatching. In C. elegans astacins, three basic structural and functional moieties can be discerned: a prepro portion, the central catalytic chain and long C-terminal extensions with presumably regulatory functions. Within the regulatory moiety, EFG-like, CUB, SXC, and TSP-1 domains can be distinguished. Based on structural differences of the regulatory unit we established six NAS subgroups, which seemingly represented different functional and evolutionary clusters. This pattern deduced exclusively from the domain arrangement in the regulatory moiety is perfectly reflected in an evolutionary tree constructed solely from amino acid sequence information of the catalytic chain. Related catalytic chains tend to have related regulatory extensions. The notable gene, NAS-39 shows a striking resemblance to human BMP-1 and the tolloids.