Freyd G et al. (1991) International C. elegans Meeting "lin -11- lacZ FUSlON GENE IS DEVELOPMENTALLY REGULATED AND EXPRESSED IN SPECIFIC VULVAL, GONADAL, AND NEURONAL CELLS."

Freyd G, Horvitz HR

[International C. elegans Meeting, 1991]

Mutations in the gene lin-ll cause the daughters of the 2 vulval precursor cells to express the same fate, producing a symmetric rather than a wild-type asymmetric lineage. Mutant animals cannot lay eggs and are slightly uncoordinated. We previously reported that lin-ll encodes a protein with both a homeodomain and two copies of a potential metal-binding motif called LIM (Nature 344: 876,1990). To understand how lin-ll functions in making the vulval 2 daughter cells different from each other, we sought to identify cells in which lin-ll is expressed. We constructed a translational fusion between genomic DNA corresponding to the upstream regulatory regions and amino terminal end of lin-l 1 and the lacZ gene on a vector obtained from Andy Fire. We coinjected into lin-ll(n389) mutant animals the lin-11- lacZ fusion with lin-ll genomic DNA and a single-stranded 50-base DNA oligomer which has been shown to increase rates of genomic integration (Mello, WBG 11:2: 28,1990). We obtained a line rescued for the Lin-ll mutant phenotype in which the lin-11- lacZ fusion plasmid had integrated into chromosome IV. We have examined expression of this fusion gene in wild-type and various mutant backgrounds. Staining in the vulval region begins during the late L3 larval stage, when the vulval precursor cells have divided twice, and largely disappears by the time the animal reaches adulthood. Staining flrst appears in two of the four descendant cells of each of the 2 vulval precursor cells. These same cells, N and T, have altered patterns of cell division in lin-ll mutant animals. Based on this observation, we propose that neither lin-ll RNA nor lin-ll protein is being asymmetrically segregated by the divisions of the vulval 2 cells. Six gonadal cells, which have been preliminarily identified as descendants of the three ventral uterine precursors, also begin staining in the late L3 larval stage. In addition, the six VC neurons in the ventral nerve cord, which innervate the vulval muscles, begin to stain late during the L3 larval stage and continue to stain throughout adulthood. Staining appears embryonically in at least nine neurons in the head and continues throughout the life of the animal. Preliminary identification by Cori Bargmann based on the location of the nuclei suggests they are the pharyngeal neuron IS, two pairs of amphid sensory neurons ADF and ADL, and two pairs of interneurons AIZ and RIC. It is possible that lin-ll expression in some of these interneurons is required for the animal to move properly, accounting for the slightly Unc phenotype of lin-ll mutants. To confirm cell identities and to examine genetic control of lin-ll expression, we are examining staining in mutants that specifically affect vulval, gonadal or VC neurons.

Zhu M et al. (2023) Chemosphere "The concentration-dependent physiological damage, oxidative stress, and DNA lesions in Caenorhabditis elegans by subacute exposure to landfill leachate."

Wang J, Zhang M, Tang M, Zhu M, Liu L, Wang Z

[Chemosphere, 2023]

The leakage of landfill leachate (LL) into environmental media would be happened even in the sanitary/controlled landfill, due to the deterioration of geomembrane and the blockage of drainage system after long-term operation. Considering the complex composition and high concentration of pollutants in LL, its toxicity assessment should be conducted as a whole liquid contaminant. Therefore, the impacts of LL on Caenorhabditis elegans (C. elegans) were investigated under the condition of different exposure time and exposure volume fraction (EVF). The stimulating effects on locomotion behavior and growth of C. elegans were observed after acute (24 h) exposure to LL, which were increased firstly and then decreased with the increase of EVF. Meanwhile, the intestinal barrier was not affected by LL, and levels of reactive oxygen species (ROS) and cell apoptosis significantly decreased. However, stimulation and inhibition effects on locomotion behavior and growth of C. elegans were observed when subacute (72 h) exposure to 0.25%-0.5% and 1%-4% of LL, respectively. The intestinal injury index and levels of ROS and cell apoptosis significantly increased when EVF were 2% and 4%. Although the acute exposure of LL had resulted in obviously biological adaptability and antioxidant defense in C. elegans, the protective mechanisms failed to be induced as the exposure time increased (subacute exposure). The toxic effects were confirmed by the down-regulation of genes associated with antioxidant defense and neurobehavior, accompanied by the up-regulation of intestinal injury and cell apoptosis related genes. Moreover, the disturbance of metabolic pathways that associated with locomotion behaviors, growth, and antioxidant defense provided good supplementary evidence for the confirmation of oxidative stress in C. elegans. The research results verified the potential of C. elegans as model organism to determine the complex toxic effects of LL.

JL Lissemore et al. (1999) International C. elegans Meeting "Using PCR in an undergraduate lab course to detect deletions in the unc-93 gene"

JL Lissemore, LL Lackner, GD Fedoriw

[International C. elegans Meeting, 1999]

PCR is a powerful and common molecular biology technique with many applications. We have developed an exercise for an undergraduate molecular biology lab course using PCR to detect deletions in the unc-93 gene of C. elegans. unc-93 encodes a putative transmembrane protein muscle protein of unknown function. Wildtype and three different unc-93 deletion mutant strains (carrying alleles lr12, lr28, and lr81) were grown, harvested, and frozen at -20 C by the instructor. Students isolate genomic DNA from each strain using a genomic DNA isolation kit from Gentra Systems, Inc. (Minneapolis, MN). We have used this kit for several years and it seems to be almost foolproof in the hands of undergraduates. Following proteinase K and RNase A treatment, proteins in the worm extract are precipitated by high salt and the genomic DNA is recovered by isopropanol precipitation. PCR reactions using two sets of primers (A and B) from two different regions of the unc-93 gene are carried out on the genomic DNA from wildtype and mutant strains and the results analyzed by agarose gel electrophoresis. Primer pair A yields a 789 bp fragment, while primer pair B yields a 728 bp fragment. The A primers detect a 173 bp deletion in lr28 and a 78 bp deletion in lr81 while the B primers detect a 517 bp deletion in lr12. The use of wildtype DNA and primers for amplifying two different regions of the unc-93 gene provide internal controls. Five student groups carried out this exercise during the Spring 1999 semester. While there was variation in the yield of the products, especially with primer pair B, all five groups detected the presence of deletions of the expected size in the expected strains. We gratefully acknowledge the assistance of Dr. Beth DeStasio, Biology Dept., Lawrence University, Appleton, WI, who provided the unc-93 strains and primers used in this exercise. LLL and GDF made equivalent contributions to this work.

Grenache DG et al. (1991) International C. elegans Meeting "GENETIC ANALYSIS OF MUTATIONS THAT ALTER TIMING OF EXPRESSION OF AN L1-SPECIFIC SURFACE ANTIGEN"

Grenache DG, Hemmer RM, Chin KJ, Politz SM

[International C. elegans Meeting, 1991]

We are using an immunogenetic approach to study genes that affect the timing of appearance of an Ll-specific cuticle surface antigen. Monoclonal antibodies M37 and M38 (class IgM) bind to the surface of live Ll wild-type animals but not to later larval stages or adults. Using indirect immunofluoresence as a phenotypic marker, we have isolated nine independent, apparently heterochronic mutants that bind M37 or M38 at developmental stages Ll-L4, but not as adults. These mutants, obtained by screening F2 progeny of EMSmutagenized N2 parents for binding of M38, have no strong visible phenotypes. The antigen recognized by M38 has been characterized in extracts of Ll cuticles by Western blotting. Antigenicity is destroyed by treatment of extracts with O-glycanase, suggesting that the antigen is an O-linked glycoprotein. The antigen has been detected in Western blots of extracts from synchronous L4 populations of one of these heterochronic mutants srf-(vj43), but no antigenicity is detectable in Western blots of corresponding extracts from wild-type L4 animals. This suggests that the srf-(vi43) phenotype is caused by heterochronic expression rather than ectopic exposure of an antigen that is normally sequestered internally. Six of the heterochronic mutations show linkage to chromosome II visible markers. Heterochronic mutant srf-( vj43) was mapped in a three-factor cross with dpy-10 and unc-4 to the left of dpy-10 or close to it on the right. srf-(yi43) complements five of the other srf mutants, suggesting that more than one gene is involved in the regulation of the developmental transition occurring at the Ll to L2 molt. In order to compare srf heterochronic phenotypes to those of previously characterized heterochronic mutants, binding of Ll-specific monoclonal to lin-4(e912), lin-29 and lin- 14(nl79)ts was tested. In contrast to the srf mutants, all of these lin mutants bound the antibody at the Ll stage only. These results suggest that the srf mutations have no effect on the larval-adult switch, and lin heterochronic mutations have no effect on the Ll-L2 stage transition.

Noordin R et al. (2004) Filaria J "Homologs of the Brugia malayi diagnostic antigen BmR1 are present in other filarial parasites but induce different humoral immune responses."

Aziz RA, Noordin R, Ravindran B

[Filaria J, 2004]

BACKGROUND: The recombinant antigen BmR1 has been extensively employed in both ELISA and immunochromatographic rapid dipstick (Brugia Rapid) formats for the specific and sensitive detection of IgG4 antibodies against the lymphatic filarial parasites Brugia malayi and Brugia timori. In sera of individuals infected with Wuchereria bancrofti the IgG4 reactivity to BmR1 is variable, and cross-reactivity of sera from individuals infected with Onchocerca volvulus or Loa loa was observed only in single cases. In order to characterize the homologs of the BmR1 antigen in W. bancrofti (Wb-BmR1), O. volvulus (Ov-BmR1) and L. loa (Ll-BmR1) the cDNA sequences were identified, the protein expressed and the antibody reactivity of patients' sera was studied. METHODS: PCR methodology was used to identify the cDNA sequences from cDNA libraries and/or genomic DNA of W. bancrofti, O. volvulus and L. loa. The clones obtained were sequenced and compared to the cDNA sequence of BmR1. Ov-BmR1 and Ll-BmR1 were expressed in E. coli and tested using an IgG4-ELISA with 262 serum samples from individuals with or without B. malayi, W. bancrofti, O. volvulus and L. loa infections or various other parasitic infections. BmR1, Ov-BmR1 and Ll-BmR1 were also tested for reactivity with the other three IgG subclasses in patients' sera. RESULTS: Wb-BmR1 was found to be identical to BmR1. Ov-BmR1 and Ll-BmR1 were found to be identical to each other and share 99.7% homology with BmR1. The pattern of IgG4 recognition of all serum samples to BmR1, Ov-BmR1 and Ll-BmR1 were identical. This included weak IgG4 reactivities demonstrated by L. loa- and O. volvulus-infected patients tested with Ov-BmR1 and Ll-BmR1 (or BmR1). With respect to reactivity to other IgG subclasses, sera from O. volvulus- and L. loa-infected patients showed positive reactions (when tested with BmR1, Ov-BmR1 or Ll-BmR1 antigens) only with IgG1. No reactivity was observed with IgG2 or with IgG3. Similarly, ELISAs to detect reactivity to other anti-filarial IgG subclasses antibodies showed that sera from individuals infected with B. malayi or W. bancrofti (active infections as well as patients with chronic disease) were positive with BmR1 only for IgG1 and were negative when tested with IgG2 and with IgG3 subclasses. CONCLUSIONS: This study demonstrates that homologs of the BmR1 antigen are present in W. bancrofti, O. volvulus and L. loa and that these antigens are highly conserved. Recognition of this antigen by patients' sera is similar with regard to IgG1, IgG2 and IgG3, but different for IgG4 antibodies. We conclude that the BmR1 antigen is suitable for detection of IgG4 antibodies in brugian filariasis. However, its homologs are not suitable for IgG4-based diagnosis of other filarial infections.

Fang K et al. (2001) J Antibiot (Tokyo) "New biotransformation products of nemadectins."

Schlingmann G, Fang K, Enos A, Carter GT

[J Antibiot (Tokyo), 2001]

Selected nemadectins (formerly LL-F28249 series) have been fed to a panel of microorganisms with the aim of generating new derivatives. In addition to products resulting from the oxidation of the terminal methyl group (C-29), a unique phosphorylated nemadectin was isolated. The phosphate group was determined to be at C-23 by HMBC between phosphorus and H-23. Milbemycin or nemadectin derivatives with natural substituents involving the 23-hydroxyl group were hitherto unknown.

Newman AP et al. (1994) Worm Breeder's Gazette "Induction of the Uterine Fate By the Anchor Cell Results in lin-11-lacZ Expression and Requires egl-29"

Sternberg PW, Newman AP

[Worm Breeder's Gazette, 1994]

Induction of the uterine P. fate by the anchor cell results in lin-ll-lacZ expression and requires egl-29. Anna P. Newman and Paul W. Sternberg, Division of Biology, 156-29, Caltech, Pasadena, CA The cells of the ventral portion of the hermaphrodite uterus arise from intermediate precursors that can have one of two fates, P. or p.. P. and p. cells differ in the morphology of their progeny and the number of descendants generated. Furthermore, as initially observed by Gwen Freyd and Bob Horvitz and confirmed by us, a lin-ll-lacZ fusion protein is specifically expressed in the P. cells and their progeny during L3 lethargus/ early IA. A signal from the anchor cell is required to induce the P. fate; ablation of the anchor cell at a time when replacement regulation can no longer occur leads to a p. --> p cell fate transformation and to an absence of lin-ll-lacZ expression in the uterus. Induction of P. fates by the anchor cell is temporally and genetically distinct from anchor cell induction of the vulva and requires lin-12 activity (WBG 12(5) p.52). Ablation of the P. cells leads to an egg-laying defect. Therefore, we screened the Class A Egl mutants of Trent, Tsung & Horvitz in order to identify additional genes required to produce P. cells. We found that egl-29(n482) has a defect in n fate specification. However, other lin-12- dependent events such as the AC vs. W decision and specification of the vulval 2 fate are wild type. Thus, egl- 29 may interact with lin-12 in a tissue-specific manner to specify the P. fate. egl-29 mutants have little uterine lin- ll-lacZ staining; staining in occasional animals probably reflects the leakiness of the allele used. An egl-29(n482);lin-12(d) double mutant has excess P. cells like a lin-12(d) mutant, suggesting that egl-29 functions upstream of lin-12. Analysis of the egl-29 null phenotype and cloning of egl-29 are in progress.

Vowels JJ et al. (1991) International C. elegans Meeting "SENSORY MECHANISMS INVOLVED IN DAUER LARVA FORMATION."

Thomas JH, Vowels JJ

[International C. elegans Meeting, 1991]

Dauer larva formation in C. elegans requires high levels of a constitutively secreted dauer pheromone. We are studying the genes and chemosensory cells involved in the transduction of the pheromone signal. Mutations in genes affecting dauer larva formation fall into two classes, the dauer constitutives (Daf-c) and the dauer defectives ( Daf-d). The Daf-c mutants form dauers in the absence of pheromone; the Daf-d mutants do not form dauers even in the presence of high levels of pheromone. Using results from both previous and recent epistasis analysis by D. Riddle and our lab respectively, these genes have been ordered into a formal genetic pathway. A subclass of the Daf-d mutants with structurally abnormal chemosensory cilia had not been positioned in the dauer pathway. Epistasis analysis between these Daf-d 'structure' mutants and the Daf-c mutants can divide the pathway into those genes acting upstream of or in the chemosensory ends and those acting downstream of the chemosensory ends. Our results suggest that the product of only one Daf-c gene, daf-ll, functions in the chemosensory ends. From a screen designed to identify Daf-d mutations acting upstream of daf-ll , we isolated one mutation, sa64, that is specifically defective in pheromone sensation; chemotaxis and avoidance responses are normal. Therefore, daf-ll and sa64 may be components of a dauer pheromone sensing mechanism. A molecular characterization of daf-ll is currently in progress. There are three classes of chemosensory cells in C. elegans; the amphids, the phasmids and the inner labial neurons. By using mutants that lack certain classes of chemosensory cells, and by laser kills of specific cells, we have been able to determine that the amphids, but not the phasmids, are necessary for sensing dauer pheromone. We also have indirect evidence that the inner labial neurons are not sufficient for dauer pheromone sensation. These results are consistent with C. Bargmann's finding that four specific amphidial neurons, ADF, ASG, ASI and ASJ regulate dauer development.

Crespo C et al. (2022) Bio Protoc "Analysis of Caenorhabditis elegans Aging-related Neurodegeneration in Chemosensory Neurons."

Crespo C, Grau R

[Bio Protoc, 2022]

Aging and neuronal deterioration constitute important risk factors for the development of neuronal-related diseases, such as different dementia. The nematode Caenorhabditis elegans has emerged as a popular model system for studying neurodegeneration diseases, due to its complete neuronal connectivity map. DiI is a red fluorescent dye that can fill the worm amphid neurons and enables the visualization of their neurodegeneration over time. This protocol provides an efficient, fast, and safe method to stain worm amphid neurons to highlight the chemosensory structures of live nematodes.

Wightman BC et al. (1991) International C. elegans Meeting "TRIGGERING OF THE lin-14 TEMPORAL SWITCH."

Gottlieb S, Wightman BC, Gatto J, Arasu P, Burglin TR, Ruvkun GB

[International C. elegans Meeting, 1991]

The heterochronic gene lin-14 controls the temporal sequence of developmental events in the postembryonic cell lineage. It encodes a nuclear protein that is normally present in most somatic cells of late embryos and Ll larvae but not in later larval stages or adults. Two lin-14 gain-of-function mutations cause an inappropriately high level of the lin-14 nuclear protein late in development but do not affect the normal temporal down regulation of transcript levels. These mutations delete 3' untranslated sequences from the lin-14 mRNAs and identify a negative posttranscriptional regulatory element that controls the formation the lin14 protein temporal gradient. The 21 kb lin-14 gene contains 13 exons that are differentially spliced to generate two distinct protein products with variable N-terminal regions and a constant C-terminus. No protein sequence similarity to any proteins in various databases was found. The normal down-regulation of lin-14 protein within 10 hours of hatching is not determined by the passage of time per se, but rather is triggered in response to feeding or initiation of postembryonic development. Animals which were suspended at Ll by starvation for up to 100 hours continued to express high levels of Zin-14 protein. Upon feeding, Zin-14 protein levels fall as postembryonic development ensues. The temporal expression pattern of lin-14 protein accumulation is regulated by the heterochronic genes lin-4 and lin-28. Iin-4 is required for the mid Ll stage down-regulation of lin-14 protein levels. In contrast, lin28 positively regulates lin-14 protein levels. In lin- 28 mutants which are arrested by starvation at Ll, lin-14 protein levels fall over time, while arrested lin-4 mutants maintain high lin- 14 protein levels, like wild-type. Either down-regulation of Zin-28 or up-regulation of Zin-4 during postembryonic development could be responsible for down-regulation of Zin14. However, since down- regulation of Zin-14 is dependent on a negative element, we postulate a model in which Zin-4 is up-regulated during postembryonic development, and subsequently interacts with the 3' untranslated region of Zin-14 message thereby mediating the downregulation of lin- 14 protein. In addition to regulation over time, different Zin-14 products may be expressed in different cell types. To address this issue, we are fusing specific lin-14 transcripts to lacZ, creating stable transformed lines, and determining the spatial and temporal expression patterns of the different products.