Blot J et al. (2002) Dev Biol "Cell cycle regulation of pEg3, a new Xenopus protein kinase of the KIN1/PAR-1/MARK family."

Chartrain I, Tassan JP, Philippe M, Blot J, Roghi C

[Dev Biol, 2002]

We report the characterization of pEg3, a Xenopus protein kinase related to members of the KIN1/PAR-1/MARK family. The founding members of this newly emerging kinase family were shown to be involved in the establishment of cell polarity and both microtubule dynamic and cytoskeleton organization. Sequence analyses suggest that pEg3 and related protein kinases in human, mouse, and Caenorhabditis elegans might constitute a distinct group in this family. pEg3 is encoded by a maternal mRNA, polyadenylated in unfertilized eggs and specifically deadenylated in embryos. In addition to an increase in expression, we have shown that pEg3 is phosphorylated during oocyte maturation. Phosphorylation of pEg3 is cell cycle dependent during Xenopus early embryogenesis and in synchronized cultured XL2 cells. In embryos, the kinase activity of pEg3 is correlated to its phosphorylation state and is maximum during mitosis. Using Xenopus egg extracts we demonstrated that phosphorylation occurs at least in the noncatalytic domain of the kinase, suggesting that this domain might be important for pEg3 function. (c)2002 Elsevier Science.

Yang J et al. (1994) Worm Breeder's Gazette "Western Blot Analyses of SQT-1 and Its Mutants"

Yang J, Kramer JM

[Worm Breeder's Gazette, 1994]

sqt-1 androl-6 encode collagens that we can now confidently call cuticle collagens EMS and in vitro o n-mutagenesis ofsqt-1 androl-6 revealed conserved amino acids in the amino and carboxyl non-Gly-X-Y domains that are important for normal function (see Worm Meeting Abstract, 1993). Mutations in carboxyl cysteines generate left roller (LRol) animals (sc13 andsc113 ).Loss of the strong positive charge at position 1 or 4 of Homology Block A (HBA R(1), V(2), R(3), R(4), Q(5)) causes a dominant right roller (RRol), recessive dumpy (Dpy) phenotype (e1350 andsc1 ).The conserved pattern of arginines of HBA suggests that HBA may be the cleavage site of a subtilisin-like endoproteinase. In order to examine mutations at the biochemical level, we have generated antipeptide antisera againstSQT-1 .On Western blots affinity purified antisera react to cuticle extracts fromN2 but notsqt-1 null mutant animals, demonstrating the specificity of the antiserum. InN2 cuticle extracts two major bands at 70 kDa and 120 kDa and some higher molecular weight material are seen to react. We believe that the 70 kDa molecule represents a dimer of two collagen chains based on the size ofSQT-1 after the predicted cleavage at HBA (25 kDa) and the fact that collagens migrate 40% slower than expected on SDS-PAGE. The two collagen chains in the dimer and the larger multimers, are presumably di/tri-tyrosine cross-linked. The bulk of cuticle collagens migrate at 90-98kDa (trimer), indicating that the cross-linking ofSqt-1 differs from the majority of collagens. Cuticle extracts fromsqt-1 mutants show differentSQT-1 patterns. The dominant RRol HBA mutants show very weak reactivity, indicating that most of these mutantSQT-1 molecules do not reach the cuticle presumably due to lack of cleavage at HBA. The LRol mutants show reduced levels of the very high molecular weight material and an increased amount of an approximately dimer size (68 kDa) molecule that runs slightly faster than in wild-type, indicating that the cysteine replacement mutations affect the formation of non-reducible crosslinks. To detectSQT-1 before it has been extensively crosslinked during cuticle assembly, we have examined extracts from molting animals 100 molting worms (mostlyL4 /Ad)were picked directly into sample buffer (containing SDS and BME) and boiled before loading. Compared to cuticle extracts, the extracts fromN2 molting worms show increased intensity of the 70 kDa dimer band and a weak band at 36 kDa, expected to be aSQT-1 monomer. Extracts of molting LRol animals allow the faster migrating (68 kDa) dimer band of reduced intensity and a very heavy 36 kDa (monomer) band. TheSQT-1 monomer (36 kDa), dimers of wild-type (70 kDa) and LRol (68 kDa) are all disulfide bonded into the cuticle since they are only solubilized in the presence of BME. The accumulation of the 36 kDaSQT-1 monomers in LRol mutants indicates that mutation of the carboxyl cysteine inhibits dimerization. Since the cysteine is immediately preceded by a conserved tyrosine, it appears that the loss of disulfide bonding inhibits the neighboring tyrosine from participating in cross-linking. We have also made antipeptide antisera against theROL-6 cuticle collagen The preliminary results show that inN2 cuticle extractsROL-6 has two major bands that are similar, but not identical in size, to theSQT-1 bands. This result indicates thatSQT-1 andROL-6 are not crosslinked together by nonreducible bonds

McGhee JD (1987) Biochemistry "Purification and characterization of a carboxylesterase from the intestine of the nematode Caenorhabditis elegans."

McGhee JD

[Biochemistry, 1987]

The major intestinal esterase from the nematode Caenorhabditis elegans has been purified to essential homogeneity. Starting from whole worms, the overall purification is 9000-fold with a 10% recovery of activity. The esterase is a single polypeptide chain of Mr 60,000 and is stoichiometrically inhibited by organophosphates. Substrate preferences and inhibition patterns classify the enzyme as a carboxylesterase (EC 3.1.1.1), but the physiological function is unknown. The sequence of 13 amino acid residues at the esterase N- terminus has been determined. This partial sequence shows a surprisingly high degree of similarity to the N-terminal sequence of two carboxylesterases recently isolated from Drosophila mojavensis [Pen, J., van Beeumen, J., & Beintema, J. J. (1986) Biochem. J. 238, 691-699].

Jeong DE et al. (2018) Methods Mol Biol "Western Blot Analysis of C. elegans Proteins."

Lee SV, Jeong DE, Lee Y

[Methods Mol Biol, 2018]

C. elegans has been widely used as a model organism for basic biological research and is particularly amenable for molecular genetic studies using a broad repertoire of techniques. Biochemical approaches, including Western blot analysis, have emerged as a powerful tool in C. elegans biology for understanding molecular mechanisms that link genotypes to phenotypes. Here, we provide a protocol for Western blot analysis using protein extracts obtained from C. elegans samples.

Egelhofer TA et al. (2011) Nat Struct Mol Biol "An assessment of histone-modification antibody quality."

Alekseyenko AA, Lieb JD, Elgin SC, Egelhofer TA, Ahringer J, Perry M, Ren B, Vielle A, Karpen GH, Schwartz YB, Gorchakov AA, Klugman S, Hawkins RD, Kolasinska-Zwierz P, Day DS, Riddle NC, Gu T, Luu Y, Ngo Q, Latorre I, Kuroda MI, Gadel S, Park PJ, Shanower GA, Linder-Basso D, Lee K, Kharchenko PV, Pirrotta V, Rechtsteiner A, Cheung MS, Kuan S, Strome S, Minoda A

[Nat Struct Mol Biol, 2011]

We have tested the specificity and utility of more than 200 antibodies raised against 57 different histone modifications in Drosophila melanogaster, Caenorhabditis elegans and human cells. Although most antibodies performed well, more than 25% failed specificity tests by dot blot or western blot. Among specific antibodies, more than 20% failed in chromatin immunoprecipitation experiments. We advise rigorous testing of histone-modification antibodies before use, and we provide a website for posting new test results (http://compbio.med.harvard.edu/antibodies/).

Murakami S et al. (1999) Curr Biol "Life extension and stress resistance in Caenorhabditis elegans modulated by the tkr-1 gene."

Johnson TE, Murakami S

[Curr Biol, 1999]

In this Brief Communication, which appeared in the 14 September 1998 issue of Current Biology, the UV dose was reported erroneously. The dose reported was 20 J/m2 but the actual dose used was 0.4 J/cm2. Also, the gene formally referred to as tkr-1 has since been renamed old-1 (overexpression longevity determination).

Ramos CG et al. (2014) J Bacteriol "Retraction for Ramos et al., MtvR is a global small noncoding regulatory RNA in Burkholderia cenocepacia."

Feliciano JR, da Costa PJ, Ramos CG, Grilo AM, Leitao JH

[J Bacteriol, 2014]

Volume 195, no. 16, p. 35143523, 2013. A number of problems related to images published in this paper have been brought to our attention. Figure 1D contains duplicated images in lanes S and LE, and Fig. 4D and 6B contain images previously published in articles in this journal and in Microbiology and Microbial Pathogenesis, i.e., the following: C. G. Ramos, S. A. Sousa, A. M. Grilo, J. R. Feliciano, and J. H. Leitao, J. Bacteriol. 193:15151526, 2011. doi:10.1128/JB.01374-11. S. A. Sousa, C. G. Ramos, L. M. Moreira, and J. H. Leitao, Microbiology 156:896908, 2010. doi:10.1099/mic.0.035139-0. C. G. Ramos, S. A. Sousa, A. M. Grilo, L. Eberl, and J. H. Leitao, Microb. Pathog. 48:168177, 2010. doi: 10.1016/j.micpath.2010.02.006. Therefore, we retract the paper. We deeply regret this situation and apologize for any inconvenience to the editors and readers of Journal of Bacteriology, Microbial Pathogenesis, and Microbiology.

Nillegoda NB et al. (2015) Nature "Crucial HSP70 co-chaperone complex unlocks metazoan protein disaggregation."

Berynskyy M, Morimoto RI, Bukau B, Stengel F, Kirstein J, Szlachcic A, Arnsburg K, Stank A, Scior A, Nillegoda NB, Gao X, Guilbride DL, Aebersold R, Wade RC, Mayer MP

[Nature, 2015]

Protein aggregates are the hallmark of stressed and ageing cells, and characterize several pathophysiological states. Healthy metazoan cells effectively eliminate intracellular protein aggregates, indicating that efficient disaggregation and/or degradation mechanisms exist. However, metazoans lack the key heat-shock protein disaggregase HSP100 of non-metazoan HSP70-dependent protein disaggregation systems, and the human HSP70 system alone, even with the crucial HSP110 nucleotide exchange factor, has poor disaggregation activity in vitro. This unresolved conundrum is central to protein quality control biology. Here we show that synergic cooperation between complexed J-protein co-chaperones of classes A and B unleashes highly efficient protein disaggregation activity in human and nematode HSP70 systems. Metazoan mixed-class J-protein complexes are transient, involve complementary charged regions conserved in the J-domains and carboxy-terminal domains of each J-protein class, and are flexible with respect to subunit composition. Complex formation allows J-proteins to initiate transient higher order chaperone structures involving HSP70 and interacting nucleotide exchange factors. A network of cooperative class A and B J-protein interactions therefore provides the metazoan HSP70 machinery with powerful, flexible, and finely regulatable disaggregase activity and a further level of regulation crucial for cellular protein quality control.

Miller DM et al. (1992) Worm Breeder's Gazette "unc-4 LacZ Expression in A-Type Motor Neurons"

Miller DM, Niemeyer CJ

[Worm Breeder's Gazette, 1992]

unc-4 LacZ expression in A-type motor neurons David M. Miller and Charles J. Niemeyer, Dept. of Cell Biology, Duke Univ. Medical Ctr, Durham, NC 27710

Sommer RJ et al. (1994) Worm Breeder's Gazette "Evolution of Vulva-Formation: Part II: Species With a Central Vulva"

Sommer RJ, Sternberg PW

[Worm Breeder's Gazette, 1994]

Evolution of vulva-formation: Part II: Species with a central vulva Ralf J. Sommer & Paul W. Sternberg, California Institute of Technology, Division of Biology 156-29, Pasadena, CA 91125