Page MF et al. (1998) Worm Breeder's Gazette "SMG-2 is a phosphoprotein"

Page MF, Anderson P

[Worm Breeder's Gazette, 1998]

Nonsense-mediated mRNA decay is a surveillance system that rapidly degrades messenger RNAs containing premature stop codons. Nonsense-mediated mRNA decay has been observed in many eukaryotes including yeast, Drosophila, humans and plants. This nonessential system of decay has been characterized genetically in C.elegans. Function of the seven smg genes is required for nonsense-mediated mRNA decay. Mutations in any of these genes abolish NMD, and nonsense mutant mRNAs accumulate to wild-type levels. smg-2 encodes a protein containing a probable C6 zinc finger domain, and a type I RNA helicase domain with a nucleotide-binding motif. We raised antibodies against the amino terminal portion of a His-tagged SMG-2, fusion protein. These antibodies detect, on a western blot, a single band of the expected 120 Kd in N2 animals. No signal is detected in smg-2 null mutants. The same single 120Kd band is found in smg-1, smg-3 and smg-4 mutants. In smg-5, smg-6 and smg-7 mutants, however, a more slowly migrating SMG-2 isoform is present in addition to the abundant 120Kd isoform. When smg-5 extracts are treated with lambda phosphatase, the more slowly migrating isoform is no longer detected, demonstrating that SMG-2 is a phosphoprotein and that the phosphorylated form accumulates in smg-5, smg-6 and smg-7 mutants. We believe that phosphorylated SMG-2 is too transient to detect in N2 worms. We have tested which smg genes are required for SMG-2 phosphorylation by double-mutant analysis. A smg mutant that accumulates the phosphorylated form of SMG-2 can be combined with a second smg mutation. If no phosphorylated form is detected in the double mutant, the second smg gene is required for SMG-2 phosphorylation. From this double mutant analysis, smg-1, smg-3 and smg-4 are required for phosphorylation of SMG-2. SMG-1 encodes a putative PI-3/protein kinase domain. We are currently testing whether SMG-1 interacts with SMG-2 and if a smg-1-dependent kinase activity coimmunoprecipates with SMG-2. The analysis of SMG-2 in smg single and double mutants divides the smg genes into two functional classes, those required to phosphorylate SMG-2 and those required for normal dephosphorylation of SMG-2. This is the first grouping of genes involved in NMD and it is hoped this will provide a framework on which to build a biochemical pathway.

Ray C et al. (1990) Worm Breeder's Gazette "CORRECTION Isolation of a C. elegans Cysteine Protease Gene"

McKerrow JH, Ray C

[Worm Breeder's Gazette, 1990]

In the previous issue of the WBG (Vol. 11, #3, page 20) we reported the isolation of a cysteine protease clone from a mixed-stage C. elegans cDNA library. This library was originally obtained from Chris Martin and not from Cynthia Kenyon as was reported in the article. Our apologies for any misunderstanding caused by this oversight.

Lewis JA (1988) Worm Breeder's Gazette "referencing, crossreferencing, and creating reference lists with Microsoft Word."

Lewis JA

[Worm Breeder's Gazette, 1988]

I've written a little primer on how to use Word (version 3.0 or higher) to automatically generate references (reference number or author-year citation) and cross-references (to page number) within a document using a combination of Word's merge function and table of contents generation facility. Also included in the article are directions for using reference information stored in a database to automatically generate a reference list according to a given style using Word's merge function. The utility of all this is likely to be superseded by future versions of Word and commercially available add- on programs. However, if you are interested, drop me a line and I'll send you a copy of my article.

Hill DP et al. (1986) Worm Breeder's Gazette "the critical times microfilaments are needed to generate asymmetries in the first cell division of C elegans."

Strome S, Hill DP

[Worm Breeder's Gazette, 1986]

Recently, we have been determining the time microfilaments (MFs) are needed to generate and maintain proper asymmetry during the first cell cycle of Caenorhabditis We can disrupt the MF system using the MF inhibitor cytochalasin D (CD). As an indication of asymmetry, we have been observing the behaviour of the zygote during pseudocleavage, pronuclear migration, segregation of germ-cell- specific granules (P granules), and first cell cleavage, all of which clearly show some indication of asymmetry. As has already been reported, when embryos are made permeable to CD early in the first cell cycle all manifestations of asymmetry are destroyed: pseudocleavage disappears, the egg and sperm yronuclei meet in the center of the embryo, P granules fail to segregate posteriorly and the first mitotic spindle sets up and remains symmetrically placed throughout karyokinesis. If one-cell embryos are treated with CD after the pronuclei have met and P granules have been segragated, P granules remain posterior but the mitotic spindle sets up and remains symmetric. This suggests that CD affects those events which have not yet taken place, but does not reverse the asymmetries already generated. It also suggests that MFs are not required to hold P granules in the posterior of the embryo once they have been segregated there. They may be anchored to non-MF components or may not be free to diffuse due to the nature of the cytoplasm. Zygotes can be pulsed with CD by introducing drug and then washing the drug out of the zygotes at specific times. Control experiments show that CD disrupts MFs within one minute of drug treatment. The minimum time zygotes were treated with drug during experiments was two minutes. Based on three types of CD pulse experiments (see diagram), it appears that MFs are not required prior to pseudocleavage and pronuclear migration but are required from pronuclear meeting until cleavage to generate proper asymmetry. l)If the embryo is treated very early, during pronuclear formation or early pseudocleavage, and the drug is washed out two minutes later, the embryo will resume pseudocleaving and behave like a normal embryo with respect to all observable aspects of asymmetry. 2)If CD is added to the embryo prior to pronuclear migration and washed out shortly after the pronuclei reach the center of the embryo, the embryo is able to recover enough to form a cleavage furrow and carry out cytokinesis. However, the proper asymmetry of the cleavage is lost. Most often the embryo divides into two equal-sized daughters, although a small percentage of the time the furrow is formed to give either a slightly larger AB-cell or a slightly larger Pcell. The P granules are not able to recover from the treatment and remain localized in the center of the embryo as they do in a continuously cytochalasin-treated embryo. 3) Preliminary results show that embryos treated shortly after pronuclear migration and P granule segregation and then washed several minutes later also undergo symmetric cleavage. However, in these embryos, P granules are segregated to the posterior cell. We conclude from these experiments that proper MF structure is needed at a critical time, during late pseudocleavage and pronuclear migration, in order for pronuclei to meet properly and P granules to segregate properly. After this time, microfilaments are needed in order to generate proper asymmetry in the first cell division itself but are dispensible with respect to events which have already occurred (P granule localization). Further experiments will be done with various cytochalasin-pulsed embryos to examine how pulses affect the developmental potential of daughter cells. {Figure 1}

Kusch M (1982) Worm Breeder's Gazette "more about L4 and adult cuticle proteins."

Kusch M

[Worm Breeder's Gazette, 1982]

SDS-PAGE of L4 and adult soluble cuticle proteins revealed that only bands E and H were present in both developmental stages (Cox et al., 1981. Dev. Biol 86: 456-470). Analysis of these proteins by 2D gels shows that 6 spots (corresponding to 3 bands on one-dimensional gels) are identical. One of the spots makes up all of the adult band E and most of the L4 band E and has an approximate pI of 4.5. Four spots compose all of band H and have pI's from 3.15-3.3. The sixth spot has a higher molecular weight than 210K and a pI of about 4. The remaining major polypeptides are different with the L4 cuticle proteins generally more basic by about 0.5-1 pH unit than the adult cuticle proteins. In addition the major L4 cuticle proteins (H is an exception) all appear as single spots on 2D gels, whereas the major adult proteins (except E) appear as multiple spots with small charge differences. A second difference between L4 and adult cuticle proteins appears when native proteins are treated with pepsin. This enzyme digests nonhelical parts of native collagens but leaves helical regions intact. Purified [35S]-labeled cuticles were extracted with - mercaptoethanol in a buffered salt solution to isolate native molecules. The extracted proteins were digested with pepsin in 0.5M acetic acid at 6 C and analyzed by SDS-PAGE and fluorography. The major labeled bands from L4 and adult cuticles (presumably polypeptides derived from collagen triple helices) had different molecular weights. Each stage has one very prominent polypeptide and 3-5 less prominent ones, some of which may have the same molecular weights in the two developmental stages.

Strome S et al. (1983) Worm Breeder's Gazette "roles of cytoskeletal elements in generation of asymmetry and segregation of P granules."

Strome S, Wood WB

[Worm Breeder's Gazette, 1983]

Between the time of fertilization and first cleavage of C. elegans zygotes, several directed movements of cellular components, which reflect the asymmetry of the zygote, occur. The zygote undergoes contractions of the anterior membrane and pseudocleavage. The egg and sperm pronuclei migrate from opposite poles of the zygote, meet in the posterior hemisphere, and then move to the center. The first mitotic spindle forms in the center of the zygote, but becomes asymmetric with respect to its position along the anterior-posterior axis and the behavior and morphology of its asters. The segregation of P granules to the posterior pole of the zygote, which occurs concurrently with pseudocleavage and pronuclear migration, provides one of the most striking manifestations of zygote asymmetry. In order to determine whether specific cytoskeletal fiber systems are involved in P-granule segregation and other early movements and whether these early events are interdependent, I have analyzed mutant embryos and wild-type embryos treated with microtubule (MT) and microfilament (MF) inhibitors. Analysis of zyg-9(b244) embryos (described in a previous newsletter) and embryos treated with low levels of MT inhibitors (which partially phenocopy the b244 defect) demonstrate that P-granule segregation is not mediated by the mitotic spindle; even though spindle orientation is altered in mutant and treated embryos, P granules nevertheless coalesce and become localized in the posterior pole. Treatment of zygotes with levels of MT inhibitors (colcemid, vinblastine, or griseofulvin) that prevent mitosis does not prevent proper P-granule segregation or pseudocleavage but does inhibit pronuclear migration. Conversely, treatment of embryos with MF inhibitors (cytochalasin D or B) inhibits P-granule segregation and pseudocleavage without preventing pronuclear migration. In addition, the resulting spindle does not become asymmetric in position or in aster behavior in cytochalasin treated zygotes. The results suggest that P-granule segregation does not require either the spindle or cytoplasmic MTs, but that this process as well as generation of other asymmetries do require cytoskeletal functions that depend on MFs. Although MT-mediated events (pronuclear migration) and MF-mediated events (pseudo-cleavage, P-granule segregation) are both normally initiated at fertilization, these two classes of processes can be uncoupled by drugs or mutation and therefore appear to be independent. Details of these experiments will appear in Cell 35, pp. 15-25. In immunoprecipitation experiments designed to identify the molecular components of P granules, one of the anti-P-granule monoclonal antibodies (K76) precipitates a 37,000-dalton polypeptide and another anti-P-granule antibody (L416) precipitates an 83,000- dalton polypeptide from [35S]-worm homogenates. In gel blot experiments both of these antibodies bind to a ~37,000-dalton polypeptide in worm and egg homogenates. However, the K76 antibody also binds a higher and lower molecular weight species as well. Further investigation of the polypeptides recognized by these antibodies, as well as two other anti-P-granule antibodies, is under way.

Strome S (1985) Worm Breeder's Gazette "visualization of microfilaments in C elegans oocytes, zygotes, and gonadal tissue."

Strome S

[Worm Breeder's Gazette, 1985]

Several intracellular motility events in the C. elegans zygote ( pseudocleavage, segregation of germ-line-specific-F granules, and generation of an asymmetric spindle) appear to depend on microfilaments (MFs). As a first step toward trying to understand the participation of MFs in these manifestations of zygotic asymmetry, I have characterized the distribution of MFs in oocytes and early embryos, using the F-actin-specific probe phalloidin and also antiactin antibodies to stain fixed specimens. In oocytes and newly fertilized zygotes, MFs are found in a uniform cortical meshwork of fine fibers and dots or foci. In the zygote, concomitant with the intracellular movements that are believed to be MF-mediated, MFs also become asymmetrically rearranged; as the zygote undergoes pseudocleavage and as P granules become localized in the posterior of the zygote, the foci of F-actin become progressively more concentrated in the anterior hemisphere. The foci remain anterior as the spindle becomes asymmetric and the zygote undergoes first mitosis, at which time fibers align circumferentially around the zygote where the cleavage furrow will form. The distribution of MFs seen by fluorescence suggests that the anterior foci of MFs may be directly responsible for the contractions of the anterior membrane seen during pseudocleavage. Contraction of MFs at the anterior end of the zygote may push or squeeze P granules posterior. The anterior MF foci may also participate in movement of the egg pronucleus toward the sperm pronucleus and then the asymmetric movements of the mitotic spindle. We are currently testing several predictions of this model. Phalloidin and anti-actin antibodies have also been used to visualize MFs in the somatic gonad. The sheath cells that surround maturing oocytes are visibly contractile and contain an unusual array of MFs; the MFs run roughly longitudinally from the loop of the gonad to the spermatheca and are interdigitated with myosin thick filaments, but not in a sarcomeric configuration. These actin and myosin filaments probably interact to cause sheath cell contraction.

Avery L (1995) Worm Breeder's Gazette "C. elegans Internet Information Servers: Status and Statistics"

Avery L

[Worm Breeder's Gazette, 1995]

As many of you know, there is a gopher server at the CGC(elegans.cbs.umn.edu) and a World-Wide-Web (WWW) server in my lab inDallas (http://eatworms.swmed.edu/) devoted to information about Celegans. These servers are heavily used.There are several other C elegans information servers, but they are notincluded here because I don't have their usage information.The table shows that, although many connections come from net-surfers whovisit once and never again (perhaps having expired from boredom), there isa large group of repeat offenders who use the servers over and over. Many, perhaps most, come from C elegans labs. (For instance,wormworld.ucsf.edu and horvitzlab3.mit.edu are pretty obvious.)My point is, if you need to reach the C elegans community, this is a goodway to do it. There is an Announcements page on both gopher and WWW. Inthe 55-day period analyzed it was read 203 times. If you have a jobopening or a conference or anything else you'd like worm people to knowabout, you probably should be advertising there. Announcements can besubmitted by e-mail to leon@eatworms.swmed.edu, and usually appear withina few hours.

Coulson AR et al. (1989) Worm Breeder's Gazette "genome map."

Ameer H, Albertson DG, Sulston JE, Coulson AR, Waterston RH, Fishpool RM

[Worm Breeder's Gazette, 1989]

The map is now widely distributed electronically (see WBG 10(3), 67), but we are once again providing a summary for the gazette in the form of an output from the routine CHPLT. Do note that this is a provisional best guess, and that some linkages may later go away: please enquire if you need to know about the status of particular areas. When you receive cosmid clones, as stabs, please IMMEDIATELY streak them out on selective medium, pick small colonies, and grow 4ml minipreps (protocol from Alan Coulson if needed). For some cosmids, larger preps are liable to yield deleted DNA. Check that cosmid DNA appears full size (runs slower than lambda on agarose gels), then freeze a sample of good cells in 20% glycerol at -70 C. MRC computer account 'ARC' does not exist; Alan and John share account JES. A database node is now open at Seattle: modem number 206-467-2957; operator Phil Meneely. The summary of clone types given on the next page may be helpful when you are deciding which clones to request for your research. To reveal the most suitable clones for microinjection, the buried clones need to be displayed by the routine CONTASS; we will help you to do this if you ask. [See Figures 1- 3]

Liu FZ et al. (1996) Worm Breeder's Gazette "MOLECULAR IDENTIFICATION OF THE CORE-ASSOCIATED PROTEINS IN C. ELEGANS THICK FILAMENTS"

Ortiz I, Epstein HF, Liu FZ, Bauer CC, Cook RG

[Worm Breeder's Gazette, 1996]

C. elegans thick filaments are complex structures consisting of multiple protein molecules. Biochemical and electron microscope studies show that the outer layer of the filaments contain myosins A and B, which are differentially localized in central and polar regions respectively. The underlying layers contain paramyosin, a homolog of myosin rod. The cores contain a second population of paramyosin (distinguishable by 2D IEF/PAGE) associated stoichiometrically with three proteins, designated as P20, P28, and P30 (Deitiker and Epstein, J. Cell Biol., 123, 303-311, 1993). Image processing and structural modeling of electron micrographs show that the cores consist of seven subfilaments (two strands of paramyosin for each) cross-linked by internal protein rings (Epstein et al., J. Struct. Biol., 115, 163-174, 1995). In order to study potential roles of P20, P28, and P30 in the assembly of thick filaments, efforts were made to purify these three proteins. Thick filaments were purified by sedimentation on 19-38% sucrose gradients and the proteins were precipitated from the filament-containing gradient fractions. The proteins were then separated on SDS-PAGE and transferred to PVDF membranes. Protein bands of interest were subjected to endoproteinase Lys-C digestion, and the resulting peptides were separated by HPLC and then microsequenced. Both P28 and P30 are novel proteins, and cosmids (T14G12 and C46G7) containing the genes encoding them have been completely sequenced by the C. Elegans Genome Project. P28 is a 201 amino acid protein that is composed of only beta sheets interspersed with turns as predicted by secondary structure programs. P30 is a 250 amino acid protein that is predicted to have both beta sheets and alpha helices. Each of the three proteins have been mapped to different chromosomes. P20, which has not been completely sequenced, is located on chromosome II near the unc-52 gene,, whereas P28 maps to the X chromosome and P30 to chromosome IV near unc-82. Anti-peptide antibodies have been raised against both P28 and P30. In preliminary experiments the antiserums have been shown to label body-wall muscle A-bands and isolated thick filaments and cores by immunofluorescence and immunoelectron microscopy. Anti-P28 has been affinity purified against its peptide antigen. The resulting antibody labels isolated cores with a paramyosin-like periodicity and appears to react along the entire length of both cores and thick filaments. These results are consistent with the predictions of the structural model of Epstein et al. (1995) for the inner core proteins. Present approaches to characterize the function of P28 include isolation of a strain with a transposon insertion in the P28 gene by R. H. A Plasterk and K. van der Linden and injection of constructs driven by the unc-54 promoter (Okkema et al., Genetics, 135, 385-404, 1993) which produce anti-sense RNA to P28 mRNA. The possible identity of P30 and unc-82 is being tested by cosmid rescue experiments. Both P28 and P30 have been expressed in E. coli as GST fusion proteins for future biochemical experiments.