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[
Worm Breeder's Gazette,
1992]
The TcA protein is probably one of the proteins essential for Tc1 transposition. In order to study the Tc1 transposition mechanism biochemically, we would like to develop an in vitro reaction system. As an initial study of this problem, we have employed TcA protein overproduced in insect cells in DNA binding experiments with different parts of the Tc1 sequence. Previous results from "South-Western blot" experiments indicated that TcA has a high non specific affinity for DNA both single stranded and double stranded (Schukkink and Plasterk, 1990, N.A.R. 18, 895-900). To further characterize specific and non specific binding of the TcA protein, gel-retardation assays were performed with a variety of Tc1 fragments and calf-thymus DNAs. These fragments, terminally labelled and incubated with nuclear protein extracts from insect cells infected either with recombinant baculovirus or with wild-type baculovirus (subsequently abbreviated as TcA- and WT- extract, respectively), were individually tested for formation of complexes with the TcA protein. Figure 1 shows the results obtained with fragments covering 300 and 170 bp from the 5' and the 3' terminus ends of the element, respectively . A slowly migrating, diffuse complex is formed upon incubation of these fragments with the TcA-extract, but not with WT-extract . The intensity of the diffuse complex increases in a dose-dependent manner with TcA protein A (Fig. 2). Such a complex is also formed between the TcA-extract and non specific target (calf-thymus), indicating a significant non-specific affinity for DNA (Fig.3).This result was already observed for other eukaryotic transposases like the one from the P element of Drosophila melanogaster (Kaufman et al. 1989, Cell 59, 359-371). To test for the specificity of the diffuse complex, unlabeled homologous and non-homologous competitor DNA fragments were included in the binding reactions with the labelled Tc1 5' terminus. Addition of an =10 to 200-fold excess of unlabeled Tc1 5' terminus fragment competes efficiently with the formation of the homologous complex (Fig.4). On the other hand the admixture of a similar excess of unlabeled calf-thymus does not interfere as well as the unlabeled Tc1 5' terminus fragment, indicating that TcA has a less good affinity for DNA other than Tc1 (Fig.4). This results indicate that, despite its relatively strong non specific affinity for DNA, the putative transposase of Tc1 exhibits a better affinity for its Tc1 specific binding sites.
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[
Worm Breeder's Gazette,
1992]
To further characterize the DNA-transposase interaction, we have studied the biochemical parameters of the binding. This was done by filtration on nitrocellulose under reduced pressure. With this system, the DNA-enzyme complex is specifically retained on the filter as preliminary experiments showed that the non specific retention of the labelled DNA alone onto the filters was < 1 %. We first incubated labelled DNA with increasing concentrations of protein in the absence (total binding) or in the presence (non specific binding, <1%) of an excess of unlabeled DNA. As shown on figure 1, Tc1 DNA binds specifically and saturably to the protein as the amount of complex formed plateaus for transposase concentrations of 100-200 nM. Protection experiments were then performed to evaluate binding at a quantitative level. These were done by incubating fixed amounts of labelled Tc1D NAand transposase with various concentrations of unlabeled Tc1 or calf thymus DNAs. Figure 2 presents the results of the specific binding obtained. Unlabeled Tc1 DNA was found able to totally inhibit the binding of [32p] Tc1 DNA to the enzyme in a dose-dependent manner [See Figure 2] K0 ,5which is the concentration of DNA which inhibits 50 % of the [32P]DNA specific binding is 3.8 nM for Tc1 .When the same type of experiment was performed with unlabeled calf thymus DNA [See Figure 2] instead of Tc1 ,we noted that increasing concentrations of this DNA also progressively prevented the binding but with a lower affinity as compared to Tc1 DNA. In this case, the K0 ,svalue was 24 nM which indicate that the ability of calf thymus DNA to bind the transposase is roughly 5 times lower as compared to Tc1 DNA in these experimental conditions. Preliminary experiments to define the kinetics of the Tc1 DNA-transposase complex revealed some interesting features. At 25 C, the association time is rather slow (T1/2 = 41 min.) but is shorter at 37 C (T1/2 = 16 min.) as presented on figure 3. Dissociation of the complex induced by adding in the incubation medium a large excess of unlabeled Tc1 was found to be slow (at 37 C, T1 /224 hours ) which indicate that the complex is very stable at this temperature. We also achieved the binding of fixed amount of purified TcA protein with increasing concentrations of [32]p labelled Tc1 DNA either in the absence or the presence of an excess of unlabeled Tc1 .Specific binding was found to be a saturable function of the concentration of the labelled DNA. As shown in figure 4, the Scatchard plot of this specific binding is not linear which indicate the existence of two families of binding sites for Tc1 DNA. The first family of sites has a higher affinity as defined by the dissociation constant, Kd, which value is 0.26 nM and a maximal binding capacity, BmaX, of 0.97 nmol/mg of protein. The second family of sites has a decreased affinity as compared to the first one as the Kd value is 5.2 nM and a Bma,, value of 4.06 nmol/mg of protein. Work is now in progress to determine if the existence of these two families of binding sites can be correlated with our results of protection experiments, where TcA would be able to recognize specific motives of Tc1 but also less conserved sequences which might be found in non-relevant DNA.
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[
International C. elegans Meeting,
1991]
To have a better understanding of the evolutionary history of mobile elements within the Nematodes, we examined the distribution and the conservation of homologues to transposable elements from Caenorhabditis elegans (Tcl, Tc2, Tc3, Tc4, TcS and FBl) in 19 nematode species belonging to the Secernentea class. Probes specific for each element were prepared and hybridized to genomic DNA. Filters were washed under conditions of increasing stringency to estimate the degree of similarity between C.elegans transposons and their homologues present in other species. Our results show that Tcl element displays a distribution restricted to the Rhabditidae family with a poor conservation. The Tc2 and FB 1 homologous elements have the same patchy distribution within the Rhabditidae family, they were only found in Caenorhabditis and in Teralorhabditis genus. The Tc3 element is widely distributed among the nematode families. The Tc3 homologous elements are present in the majority of the Rhabditidae genera but also in the two genera of the Panagrolaimidae family, and in the Bursaphelenchus genus which belong to the Aphelenchida order. Tc4 and Tc5 homologues show the most limited distribution of all tested elements, being strictly limited to the C.elegans species. These data indicate that in some cases, the distribution of transposable elements in the nematode cannot be explained by strict vertical transmission. The distribution of Tc3, Tc4 and TcS suggests that an horizontal transmission may have occured between reproductively isolated species during their evolutionary history.
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[
J Mol Evol,
1991]
To have a better understanding of the evolutionary history of mobile elements within the nematodes, we examined the distribution and the conservation of homologues to transposable elements from Caenorhabditis elegans (Tc1, Tc2, Tc3, Tc4, Tc5, and FB1) in 19 nematode species belonging to the class Secernentea. Our results show that Tc1 elements display a distribution restricted to the family Rhabditidae with poor conservation. The Tc2 and FB1 homologous elements have the same patchy distribution within the Rhabditidae. They were only found in Caenorhabditis and in Teratorhabditis. The Tc3 element is widely distributed among nematode species. Tc3 homologous elements are present in the majority of the Rhabditidae but also in two genera within the family Panagrolaimidae, and in Bursaphelenchus, which belongs to the order Aphelenchida. Tc4 and Tc5 homologues show the most limited distribution of all tested elements, being strictly limited to C. elegans. These data indicate that in some cases, the distribution of transposable elements in the nematode cannot be explained by strict vertical transmission. The distribution of Tc3, Tc4, and Tc5 suggests that horizontal transmission may have occurred between reproductively isolated species during their evolutionary history.
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[
Biochem Biophys Res Commun,
1993]
The TcA protein is one of the proteins essential for Tc1 transposition. In order to study the biochemical parameters of Tc1 transposition mechanism, we used TcA protein overproduced in baculovirus system for DNA binding experiments. We show that, despite its relatively strong non specific affinity for DNA, TcA exhibits a better affinity for its Tc1 specific binding sites. The K0.5 is 3.8 nM for the Tc1 whereas in the same type of experiment the K0.5 is 24 nM for calf thymus DNA. The ratio value between specific and non specific DNA binding activity of the TcA protein was also exhibited by other transposases such as those of the bacteriophage Mu, Tn 10 and the Drosophila P element. This nonspecific DNA binding activity may be involved in determining sites of transposable element insertion.
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Pennington PR, Heistad RM, Nyarko JNK, Barnes JR, Bolanos MAC, Parsons MP, Knudsen KJ, De Carvalho CE, Leary SC, Mousseau DD, Buttigieg J, Maley JM, Quartey MO
[
Sci Rep,
2021]
The pool of -Amyloid (A) length variants detected in preclinical and clinical Alzheimer disease (AD) samples suggests a diversity of roles for A peptides. We examined how a naturally occurring variant, e.g. A(1-38), interacts with the AD-related variant, A(1-42), and the predominant physiological variant, A(1-40). Atomic force microscopy, Thioflavin T fluorescence, circular dichroism, dynamic light scattering, and surface plasmon resonance reveal that A(1-38) interacts differently with A(1-40) and A(1-42) and, in general, A(1-38) interferes with the conversion of A(1-42) to a -sheet-rich aggregate. Functionally, A(1-38) reverses the negative impact of A(1-42) on long-term potentiation in acute hippocampal slices and on membrane conductance in primary neurons, and mitigates an A(1-42) phenotype in Caenorhabditis elegans. A(1-38) also reverses any loss of MTT conversion induced by A(1-40) and A(1-42) in HT-22 hippocampal neurons and APOE 4-positive human fibroblasts, although the combination of A(1-38) and A(1-42) inhibits MTT conversion in APOE 4-negative fibroblasts. A greater ratio of soluble A(1-42)/A(1-38) [and A(1-42)/A(1-40)] in autopsied brain extracts correlates with an earlier age-at-death in males (but not females) with a diagnosis of AD. These results suggest that A(1-38) is capable of physically counteracting, potentially in a sex-dependent manner, the neuropathological effects of the AD-relevant A(1-42).
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[
Worm Breeder's Gazette,
2003]
Wormgenes is a new resource for C.elegans offering a detailed summary about each gene and a powerful query system.
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[
Front Pharmacol,
2020]
Oligomeric assembly of Amyloid- (A) is the main toxic species that contribute to early cognitive impairment in Alzheimer's patients. Therefore, drugs that reduce the formation of A oligomers could halt the disease progression. In this study, by using transgenic <i>Caenorhabditis elegans</i> model of Alzheimer's disease, we investigated the effects of frondoside A, a well-known sea cucumber <i>Cucumaria frondosa</i> saponin with anti-cancer activity, on A aggregation and proteotoxicity. The results showed that frondoside A at a low concentration of 1 M significantly delayed the worm paralysis caused by A aggregation as compared with control group. In addition, the number of A plaque deposits in transgenic worm tissues was significantly decreased. Frondoside A was more effective in these activities than ginsenoside-Rg3, a comparable ginseng saponin. Immunoblot analysis revealed that the level of small oligomers as well as various high molecular weights of A species in the transgenic <i>C. elegans</i> were significantly reduced upon treatment with frondoside A, whereas the level of A monomers was not altered. This suggested that frondoside A may primarily reduce the level of small oligomeric forms, the most toxic species of A. Frondoside A also protected the worms from oxidative stress and rescued chemotaxis dysfunction in a transgenic strain whose neurons express A. Taken together, these data suggested that low dose of frondoside A could protect against A-induced toxicity by primarily suppressing the formation of A oligomers. Thus, the molecular mechanism of how frondoside A exerts its anti-A aggregation should be studied and elucidated in the future.
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[
International Journal of Developmental Biology,
1998]
Pleiotropy , a situation in which a single gene influences multiple phenotypic tra its, can arise in a variety of ways. This paper discusses possible underlying mechanisms and proposes a classification of the various phenomena involved.
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[
Curr Biol,
2011]
Recent work on a Caenorhabditis elegans transmembrane ATPase reveals a central role for the aminophospholipid phosphatidylethanolamine in the production of a class of extracellular vesicles.