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[
International Worm Meeting,
2021]
The WD-40 repeat protein CDC-20 is the co-activator of the Anaphase Promoting Complex/Cyclosome (APC/C), the E3 ubiquitin ligase responsible for mitotic exit, as well as a core subunit of the mitotic checkpoint complex that restrains APC/C activity when chromosomes are not yet attached to the mitotic spindle. CDC-20 rapidly fluxes through the kinetochore region of chromosomes via association with a conserved binding motif, known as the ABBA motif, in BUB-1. CDC-20 flux through kinetochores is essential for mitotic checkpoint activation, which delays anaphase onset until all chromosomes attach to microtubules, and for promoting anaphase onset following kinetochore-microtubule attachment (Kim and Lara-Gonzalez et al, 2017, Genes and Development 31:1089-1094). Here, we investigate the regulation of BUB-1-dependent recruitment of CDC-20 to kinetochores. We found that mutating a conserved Polo-like Kinase 1 (PLK-1) docking site in BUB-1 eliminated CDC-20 kinetochore recruitment to the same extent as mutating the ABBA motif; in addition, the peak kinetochore recruitment of the mutant BUB-1 was reduced to ~50% of wildtype levels, likely due to a role for BUB-1-bound PLK-1 in promoting kinetochore recruitment of BUB-1. To address if the defect in CDC-20 kinetochore recruitment was a consequence of reduced BUB-1 kinetochore localization or was due to BUB-1-docked PLK-1 regulating the ABBA motif-CDC-20 interaction, we selectively mutated the BUB-1-associated protein BUB-3 to impair its recognition of the phosphorylated kinetochore scaffold KNL-1. The BUB-3 phospho-recognition mutant reduced peak BUB-1 kinetochore recruitment to ~30% of wildtype levels; however the consequences on anaphase onset and CDC-20 kinetochore localization were significantly less severe than observed for PLK-1-docking mutant BUB-1. These observations support a model in which BUB-1-associated PLK-1 is essential for CDC-20 kinetochore recruitment, either by direct phospho-regulation of the ABBA motif or by controlling access of the ABBA motif to CDC-20. Our current experiments are focused on distinguishing between these two potential mechanisms.
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[
Biol Pharm Bull,
2011]
We examined the sugar-cleaving abilities of -galactosidases from jack bean and Streptococcus towards sugars containing fucose residues, and found that jack bean -galactosidase has an ability to cleave the 1-3 linkage between galactose (Gal) and fucose (Fuc) residues, but not 1-4 linkage. On the other hand, streptococcal -galactosidase was found to cleave the linkage in both Gal1-4Fuc and Gal1-3Fuc disaccharide units. Such a difference in sugar-cleaving abilities between these 2 -galactosidases will be useful for structural analysis of glycans, especially those from species belonging to Protostomia, such as Caenorhabditis elegans.
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[
Worm Breeder's Gazette,
1976]
HOUSTON, July 26--Just as Cervantes and Shakespeare said it would, the once trod-upon worm has turned. Like an underground movie starlet, the worm has been transformed from rural hick into celebrity chic by shrewd promotion, abundant potential and, recently, by famous-name identification.
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[
Worm Breeder's Gazette,
1986]
We are interested in cellular and nuclear interspatial relationships. These can be delineated after three-dimensional reconstruction from serial ultrathin sections. To this end we have concocted 'Metamorphosis' which is a group of computer programs designed to permit three-dimensional analysis of objects from digitized data. Infinite exterior views are possible using the rotation program and the user has the added ability to enter the object and peer out. This permits analysis of structural associations from any perspective. Since many associations cannot be abstracted without a computer, using rotation analysis may reveal obscure relationships. This concept becomes important when proposing theories involving interactions between two or more objects. The program is written in BASICA and designed for use on IBM-compatible personal computers equipped with a digitizer. We are currently using an IBM-XT with a 10-megabyte hard disk and a Houston Instrument DT114 HIPAD Digitizer. Three dimensional transformations are accomplished via matrix manipulation which permits rapid evolution of new images. [See Figure 1]
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[
Medicines (Basel),
2016]
Murraya paniculata (L.) Jack, a small tropical evergreen shrub growing in Nepal, has numerous uses in traditional medicine for treatment of abdominal pain, diarrhea, stomach ache, headache, edema, thrombosis, and blood stasis. The present study investigated the chemical composition and bioactivities of the leaf essential oil from M. paniculata from Nepal. The essential oil from leaves was obtained by hydrodistillation and a detailed chemical analysis was conducted by gas chromatography-mass spectrometry (GC-MS). The essential oil was screened for antimicrobial activity using the microbroth dilution test, for nematicidal activity against Caenorhabditis elegans, and for lethality against brine shrimp (Artemia salina). A total of 76 volatile components were identified from the essential oil. The major components were methyl palmitate (11.1%), isospathulenol (9.4%), (E,E)-geranyl linalool (5.3%), benzyl benzoate (4.2%), selin-6-en-4-ol (4.0%), -caryophyllene (4.0%), germacrene B (3.6%), germacrene D (3.4%), and -elemene (3.2%). The essential oil showed no antibacterial activity, marginal antifungal activity against Aspergillus niger (MIC = 313 g/mL), a moderate activity against A. salina (LC50 = 41 g/mL), and a good nematicidal activity against C. elegans (LC50 = 37 g/mL).
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[
Worm Breeder's Gazette,
1980]
We have now completed the DNA terminal phenotypes for the B set of embryonic lethals of C. elegans. Flow cytometry was used to measure the total DNA content of 18 embryonic lethals maintained at restrictive temperature (25 C) for 20 hours. The enclosed Table provides these DNA terminal phenotypes as well as three (63'0', 44T, 11Y) from our Houston set of conditional embryonic lethals. The number of independent separate determinations is shown in parenthesis and the total number of embryos analyzed per sample ranged from 3,000 to 270,000 (the average is about 20K to 100K). Cell stages (or nuclear DNA equivalents) that contain more than 8% of the total number of embryos are blocked out in this Table. The above DNA terminal phenotypes are now being complemented by the direct embryonic squash technique. The results that we have obtained are: (1) the average DNA content observed by flow cytometry is reflected by a similar average number of nuclei for several mutants; ( 2) several embryonic lethals give rise to abnormally enlarged ( polyploid) nuclei, and (3) several mutants demonstrate that cleavage is independent of nuclear division. They exhibit blastomeres that do not contain nuclei. [See Figure 1]
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Pham, Kelvin, Henderson, Jacklyn, Soebianto, Sarita, Mathai, Kristina, Taylor, Remington, Nguyen, Gina, LaForce, Michael, Simmons, Dr. Alexandra
[
International Worm Meeting,
2021]
Caenorhabditis elegans is a valued model organism that is easy to maintain in the lab. We obtained a mutant strain of C. elegans that exhibits constant involuntary muscle spasms, a phenotype that has not been described before in the literature (MCW230, curtesy of Dr Wang, BCM, Houston, TX). The 'trembler' mutant strain has a
nre-1(
hd20)
lin-15b(
hd126) genetic background. We use snip-SNP mapping to seek the causal mutant gene responsible for the trembling phenotype in the MCW230 strain. MCW230 worms were backcrossed with wild-type N2 worms to eliminate the double mutant background (
nre-1,
lin-15b). For snip-SNP gross mapping, we crossed our mutant with CB4856 worms. DNA was extracted from 43 F2 individuals resulting from the MCW230 x CB4856 cross. The F2 samples were studied to identify the presence of specific single nucleotide polymorphisms at 18 genomic locations (3 per chromosome) and assess how the alleles segregate relative to the trembler phenotype. We completed genotyping at all genomic locations and our data supports the idea that the trembler phenotype seems to be linked to one of the markers on chromosome III. Our work is an example of how research using C. elegans can be done in the undergraduate environment with basic laboratory equipment.
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[
Worm Breeder's Gazette,
1980]
We are investigating somatic loss of X-chromosomes as a means of producing mosaic worms at relatively high frequencies. We produce worms heterozygous for the X-linked muscle morphology mutant,
unc-78(
e1217), which are homozygous for various him's. Only one such mutant,
him-2(
e1065), generates spontaneous mosaics at a frequency greater than 0.1%. We are still examining two other lines and are exploring other means of inducing high-frequencies of X-linked mosaics . We have observed loss of X-chromosomes from worms irradiated by X- rays from 137Cs. The loss is seen in the F2 of irradiated worms (or the F1 or irradiated embryos). The number of males in this generation is in the range of 4-10%. The frequency of males is not distributed according to a Poisson but rather shows 'jack-pots' as might be expected from events that hit individual X chromosomes. The frequency of males in a jackpot can be as high as 40%. Most hermaphrodite siblings of these males are normal and show no increase in the frequency of male progeny. We asked whether only the irradiated chromosome was lost in an animal heterozygous for a marked irradiated X-chromosome and an unmarked unirradiated X-chromosome. Hermaphrodites marked with the X- linked marker,
unc-78, were irradiated and then mated to N2 males. Wild type progeny were picked, cloned and scored for male offspring. Both wild type and
unc-78 males were found at approximately equal frequency; apparently either chromosome can be lost.
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[
European Worm Meeting,
2004]
Caenorhabditis elegans has been found to be a good model system for parasitic nematodes, drug screening and developmental studies. Structural analyses have revealed nematode specific glycosphingolipid structures of the arthro-series, carrying, in part, phosphorylcholine (PC) substituents. PC is a widespread antigenic epitope of pathogens like parasitic nematodes and has also been detected on N-glycans of this model organism (1, 2). The PC modification seems to play an important role in nematodes development, fertility and survival within the host. With the exception of ES-62 from Achanthocheilonema viteae no protein carrying this epitope has been yet identified and further characterized (3). In the axenic medium of C. elegans culture we detected a single protein with an apparent molecular mass of 40 kDa which reacted with the PC-specific antibody TEPC-15. The protein was purified by anion-exchange chromatography and 2D-gel electrophoresis. After in-gel digestion with trypsin, the protein was identified by MALDI-TOF-MS peptide finger print and nanoLC-ESI-MS/MS microsequencing as the aspartyl protease ASP-6. RNAi experiments confirmed is assignment. Lectin analysis of the purified ASP-6 protein revealed the presence of the GlcNAc-residues by binding of WGA, whereas Con A showed no binding, indicating the absence of terminal mannosyl residues. Treatment of the protein with PNGase A and PNGase F abolished the binding of WGA, but not that of TEPC-15. This might be an indication for a PC-epitope distinct from those described so far. References: 1. Lochnit, G. et al. (2000) Biol. Chem. 381: 839-47 2. Houston, K., et al. (2002) Mol. Biochem. Parasitol. 123: 55-66 3. Harnett, W., et al. (2003) Curr.Protein Pept. Sci. 4: 59-71
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[
Worm Breeder's Gazette,
1985]
Strain requests: Please remember to request strains by letter, even if you've made a request by phone. Letters should list what strains you want (strain name or gene name is fine), briefly state what you want them for, and state your funding source (no numbers, just the institution or agency). Bibliography: The complete CGC bibliography (on paper) will shortly be mailed to each lab with a CGC laboratory designation. In addition, the bibliography is now available on computer diskette in a variety of formats, including dBase III, IBM-compatible ASCII, Apple II- and Macintosh-compatible ASCII. The ASCII data can be read with line editors and many word processors. To obtain a copy, send me a blank diskette (or diskettes) formatted with PC-DOS, MS-DOS or Apple DOS and tell me which format you want. The bibliography is about 300 kilobytes in size, so send enough diskettes to contain it all. For IBM-type drives, we can handle 1.2 Mb or 360 Kb diskettes. Complete file structure information will accompany each disk sent out. Films: The CGC now has a copy of the Encyclopaedia Britannica film 'Nematode' and a copy of Einhard Schierenberg's embryonic development film. We will loan these films for a period of two weeks to any laboratory with a CGC lab designation. Requests for such loans should be made by letter from the laboratory head to me or Don Riddle at the CGC. Electronic Mail: The University of Missouri is a node of the BITNET computer network, with connections (gateways) to the CSNET, CCNET, UUCP and MAILNET networks. People with local access to BITNET can send electronic mail to the CGC by directing it to 'BIOSCGC at UMCVMB'. Communicating over gateways to BITNET requires a little more complicated addressing. Anyone who wants to try electronic mail should contact me by regular mail first for detailed instructions, so I'll know if the message gets lost. According to our BITNET node list, the following worm-breeder institutions have direct access to BITNET: Cornell University, Columbia University, University of California at Berkeley, MIT, Harvard, University of Illinois at Chicago, North Carolina State University, Washington University, University of Houston, University of Wisconsin-Madison, University of Massachusetts, SUNY at Buffalo, University of California at Santa Cruz, Duke University, University of Texas at El Paso. There are certainly other BITNET nodes that are not on our list. In Europe, EARNET can be used to connect to BITNET.