Brandon Barker et al. (2008) C.elegans Aging, Stress, Pathogenesis, and Heterochrony Meeting "Identifying regulatory elements, gene boundaries, and an analysis of gene regulation complexity"

Brandon Barker, Jim Lund

[C.elegans Aging, Stress, Pathogenesis, and Heterochrony Meeting, 2008]

With multiple genomes related to each metazoan model organism being sequenced promoter analysis is becoming an especially useful tool in genomic analysis. Comparative sequence analysis can identify functional sequence elements for pairs of organisms that diverged far enough in the past to allow mutational drift of non-conserved sequences. While analysis of a pair of genomes identifies many of these functional elements adding additional genomes allows additional information to be elicited. Additional conserved sequence elements are identified as additional genomes are analyzed. These conserved sequence elements are often regulatory elements although they are difficult to classify with in silico analysis. For an individual gene, the set of associated conserved sequence elements and the organisms they are found in provides insight into the evolutionary history of the regulation of the gene. The eight complete and unfinished shotgun sequenced nematode genomes and the dozen informative insect genome sequences were used to analyze conserved non-coding sequences in these groups of organisms. Web-based software was developed to allow researchers to explore and visualize sequence conservation that expands upon previous work by analyzing conserved sequences in each pair of organisms rather than with respect to a single reference genome. We have used this analysis to identify gene regulatory boundaries in the nematode C. elegans. The genomes of C. elegans and other nematodes have diverged enough that syntenic regions are typically a few genes long. I was able to associate conserved sequence elements to particular genes identifying the natural boundaries between genes and the extent of worm promoters. We have used the set of identified C. elegans and C. briggsae conserved promoter elements construct a promoter complexity score. Promoter complexity identifies which genes have particularly interesting regulation, identifying gene groups with a strong promoter complexity signal and cases where a gene''s promoter complexity differs from the group''s promoter complexity. Monte Carlo random sampling was used to identify Gene Ontology and KEGG Pathway annotated gene groups that appear to have significantly low or high complexity. Genes annotated as developmental genes and signaling genes especially G-protein coupled receptors and cell-cell signaling genes have high promoter complexity scores. We also examined gene expression in the published C. elegans microarray experiments and found a strong positive correlation between gene expression variation and promoter complexity. Genes showing considerable regulation in microarray experiments tend to have complex promoters.

Barker, Brandon et al. (2009) International Worm Meeting "Nematode genomic analysis identifies conserved regulatory sequences and gene boundaries."

Barker, Brandon, Lund, Jim

[International Worm Meeting, 2009]

Ten nematode genomes have been sequenced, two completed and the other eight shotgun sequenced and assembled, representing four of the twelve nematode clades. We have used comparative sequence analysis to identify conserved non-coding sequences and their distribution among these nematodes. Comparative sequence analysis can identify functional sequence elements by seqeunce conservation. While analysis of a pair of genomes identifies many of these functional elements, additional conserved sequence elements are identified as additional genomes are analyzed and the species range of an element can be used to estimate its age. These conserved sequence elements are often regulatory elements although they are difficult to classify with in silico analysis. Analysis of the four sequenced Caenorhabditis species identifies extensive conserved non-coding sequence, approximately double the conserved sequence identified comparing a pair of species. P. pacificus and H. glycines were found to have few putative promoter sequence elements conserved with C. elegans, much less than expected based on gene conservation indicating that gene regulation in these species has diverged considerably. In contrast, promoter conservation with M. hapla is more extensive than expected. We have developed web-based software to allow researchers to explore and visualize sequence conservation in the analyzed nematode genomes (http://daf.uky.edu/id_plot/). Gene regulatory boundaries in the nematode C. elegans were identified by examining evolutionary chromosomal recombination events. Conserved sequence elements were linked with particular genes identifying the natural boundaries between genes and the extent of worm promoters. We have used the set of identified C. elegans and C. briggsae conserved promoter elements to quantify and analyze promoter complexity. Monte Carlo sampling was used to identify GO and KEGG annotated gene groups that appear to have significantly low or high promoter complexity. Genes annotated as developmental genes and signaling genes especially G-protein coupled receptors and cell-cell signalling genes have high promoter complexity scores. Gene expression in the complete set of published C. elegans microarray experiments was analyzed and a strong positive correlation between gene expression variation and promoter complexity was discovered. Genes showing considerable regulation in microarray experiments tend to have complex promoters.

JK6011

Caenorhabditis elegans

(2021) Development "The people behind the papers - Brandon Carpenter and David Katz."

[Development, 2021]

A dynamic pattern of histone methylation and demethylation controls gene expression during development, with some processes such as formation of the zygote involving large-scale reprogramming of methylation states. A new paper in Development investigates how inherited histone methylation regulates developmental timing and the germline/soma distinction in <i>Caenorhabditis elegans</i> To hear more about the story we caught up with first author and postdoctoral researcher Brandon Carpenter, and his supervisor David Katz, Associate Professor in the Department of Cell Biology at Emory University School of Medicine in Atlanta, Georgia.

Asher D Cutter (2003) International Worm Meeting "How many sperm are enough?"

Asher D Cutter

[International Worm Meeting, 2003]

Sperm limited fecundity in selfing C. elegans hermaphrodites has been explained to be the consequence of a trade-off between selection for greater total fecundity and more rapid turnover of generations. Greater numbers of sperm increase the total number of progeny produced by a hermaphrodite, but delay the onset of fertilization and therefore reduce the intrinsic rate of population growth. Both empirical and theoretical work support this general explanation (Hodgkin & Barnes 1991; Barker 1992). However, the question remains: can this simple model explain the actual numbers of sperm produced by C. elegans hermaphrodites? In this study, I extend a well-known population model to calculate the expected number of sperm that hermaphrodites would produce to maximize fitness (i.e. the intrinsic rate of growth). I apply literature-based and new empirical estimates of rates of gamete production and the fraction of sperm produced during larval development to the model. I find that the model predicts sperm counts consistent with observed sperm numbers in C. elegans hermaphrodites, given available empirical data, and provided that precocious larval production of sperm is taken into account. Several testable hypotheses follow from the model regarding how natural selection and environmental variation may influence sperm production among populations or among species with a similar mode of reproduction. J. Hodgkin & T.M. Barnes. 1991. Proc. R. Soc. London B. 246: 19-24; D.M. Barker. 1992. Evolution. 46: 1951-1955.

(1990) Worm Breeder's Gazette "CGC Bibliography in FileMaker and HyperCard (Mac) and Memory Mate ( IBM)"

[Worm Breeder's Gazette, 1990]

The CGC Bibliography has been translated into a couple of programs other than dBase by various worm people. David Barker and Andy Fire both have sent HyperCard versions to the CGC and Mark Blaxter has sent us a version in FileMaker 2.0. None of these is perfectly up-to-date, so you'll have to be somewhat familiar with the programs to add new references. The data files are available free from the CGC; to get yours, just send a blank 3.5' diskette to Mark Edgley at the CGC with a request letter. In addition, Lew Jacobson has translated the bibliography into a DOS program called Memory Mate and he is willing to distribute the data file to anyone who sends him a blank 5.25' 360 Kb diskette. Memory Mate can be operated as a TSR and called up with a hotkey from the middle of a word processor or other program. Addresses for Mark and Lew can be found in the Subscriber Directory Update in this issue.

Michael Palopoli et al. (2007) International Worm Meeting "Polymorphism at the Caenorhabditis elegans plg-1 locus is due to a retrotransposon insertion that blocks transcription of a mucin-like protein."

TinMaung Aye, Camden Ramsay, Michael Palopoli, Matthew Rockman, Curwen Stephen, Jason Laurita, Leonid Krugylak

[International Worm Meeting, 2007]

Natural populations of C. elegans are polymorphic for the ability of males to deposit a copulatory plug after mating. Plugs have a mate-guarding function in this species (Barker 1994, Animal Behaviour 48:147), and the polymorphism is due to allelic variation at the plg-1 locus (Hodgkin and Doniach 1997, Genetics 146:149). We hypothesized that the polymorphism is caused by a loss-of-function mutation because (1) plugging is likely to be ancestral, since plugs have been observed in other Caenorhabditis species; (2) the plugging allele is dominant; and (3) males are rare in natural populations, so a mate-guarding function is probably dispensable. To test this hypothesis, we have identified the molecular basis for the plg-1 polymorphism. We narrowed the location of plg-1 to a 76-kb interval using a combination of recombination and physical-mapping experiments. We then discovered that natural populations of C. elegans are polymorphic for the presence of a retrotransposon insertion (Cer-1) within this genomic interval. Interestingly, this retrotransposon is inserted within genomic sequence that is predicted to code for a mucin-like protein. Furthermore, the insertion is present in all of 45 non-plugging isolates, but absent in all of 92 plugging isolates that we tested. RT-PCR experiments showed that the mucin-like gene is transcribed in adult males, but not in adult hermaphrodites, of plugging strains. In contrast, the transcript was not observed in non-plugging strains, suggesting that the retrotransposon blocks transcription of the mucin-like protein. The results of RNAi knockdown and transgenic rescue experiments confirmed the identity of the mucin-like protein as the plg-1 gene. The plg-1 polymorphism is caused by a naturally segregating retrotransposon insertion that disrupts production of a structural component of the copulatory plug.

Richer J et al. (1993) Worm Breeder's Gazette "Search for EcR Homologs in Nematodes"

Richer J, Hough DM, Maina CV

[Worm Breeder's Gazette, 1993]

Filariasis is a parasitic disease infecting over 300 million people worldwide resulting in Iymphoedema, elephantiasis, or blindness. The disease is caused by several species of helminthic worms (Brugia malayi, B. timori, Wuchereria bancrofti and Onchocerca volvulus) transmitted by mosquitoes and flies. A related disease, dog heartworm is caused by Dirofilaria immitis infection in the heart and lungs of dogs. The role of ecdysone during insect metamorphosis and the superficial similarity between development of insects and nematodes through a series of larval molts to the adult, has led to the theory that molting in nematodes may also be controlled by steroid hormone(s). The presence of ecdysone has been reported in D. immitis, (Cleator et al. 1987, Mol Biochem Parasitol 25:93-105) and ecdysone has been shown to stimulate formation of microfilaria and induce the third- to fourth-stage molt in vitro (Barker et al. 1990, Invertebrate Reprod Devel 18:1-11; Barker et al. 1991, Parasitol Res 77: 65-71). However, attempts to elucidate a biosynthetic pathway for ecdysteroids in D. immitis (Mercer et al. 1989, Trop Med Parasitol 40:429-433), C. elegans (Chitwood et al. 1990, J Nematology 22 :598-607) and other nematodes have been unsuccessful. If ecdysone is being utilized in nematodes, the receptor for the hormone will be present. The Drosophila ecdysone receptor (EcR) is a member of the nuclear hormone receptor (NHR) superfamily, a group of hormone regulated transcription factors, and has been cloned and sequenced (Koelle et al. 1991, Cell 67:59-77). We have used the Drosophila EcR to search for D. immitis and C. elegans homologs using several approaches, the most fruitful being PCR with degenerate primers to the DNA binding domain. Four degenerate primers (C1, C2 ,C4 ,and C5 )encoding the amino acids shown in the figure were used in various combinations in PCR experiments with genomic DNA from both D. immitis and C. elegans (see figure below and expanded figure in abstract by Hough et al., this issue). All PCR products were cloned into pUC19 and sequenced. PCR with primer combination C2 /C4amplified a DNA fragment of 473 bps from D. immitis (dirf-3) that upon sequencing shows 90% similarity at the amino acid level to the Drosophila EcR. It contains a putative intron of 362 bps at the position indicated by the arrow which is within the C1 primer and is most likely why it was not amplified with the C1 /C2primer combination (see below). The Drosophila EcR does not contain an intron within the DNA binding domain. Primer combination C4 /C5amplified a single DNA fragment of 455 bps from D. immitis that upon sequencing showed it to be a smaller piece of dirf-3. The C1 /C2primer combination amplified 3 DNA fragments from C. elegans (crf-3 ,crf-4 ,and crf-5 ,in keeping with the nomenclature for nematode NHR s established by Sluder et al. WBG 11, #3) and 2 from D. immitis (dirf-1 and dirf-2), ranging in size from 160 to 524 bps. All 5 show significant similarity to the NHR superfamily at the amino acid level, although no striking similarity to any one particular member. Four contain putative introns of sizes ranging from 45 to 258 bps at the sites indicated by the arrow. None of the 3 C. elegans NHR s have been described previously. They have been mapped to the genome (see figure) and we have obtained cDNA s for each from a screen of a mixed stage cDNA library (Stratagene). The 3 D. immitis NHR s are the first to be described for this nematode and, to date, we have obtained a cDNA for one (dirf-2) from a screen of an adult female cDNA library (Grandea et al. 1989, Mol Biochem Parasitol 35:31-42). Our search for a C. elegans EcR homolog continues.

Lee JH et al. (1994) Worm Breeder's Gazette "Molecular Genetics of SNAP-25 in C. elegans."

Meyer BJ, Lee JH

[Worm Breeder's Gazette, 1994]

Molecular genetics of SNAP-25 in C. elegans Junho Lee and Barbara Meyer Department of Molecular and Cell Biology 401 Barker Hall, University of California at Berkeley Berkeley, CA 94720 leejh@mendel.berkeley.edu Recent discoveries of components used in various types of membrane fusion events, from neurotransmitter release in mammalian neuronal cells to protein secretion in yeast cells, have led to a unified hypothesis. In order to extend our understanding of the biology of vesicle trafficking and membrane fusion events, it is important to study the roles of all the components of the fusion machinery. SNAP 25 is one of the components of the membrane fusion apparatus, and its molecular and biological functions have not been fully determined. It has been proposed that SNAP-25 is important in axonal growth during neural development and that it acts as a plasma membrane receptor in vesicle docking and fusion. Because this protein was shown to be conserved during evolution, it would be useful to investigate the molecular and biological functions of the SNAP-25 protein in a simpler model system such as the nematode C. elegans in order to test these hypotheses. We cloned a homolog of SNAP-25 by PCR using a set of degenerate primers that had been successfully used by C. Risinger and D. Larhammer to clone Drosopl2ila SNAP-25 homologs. They kindly provided the primers to us. We subcloned PCR-amplified products in a pBluescript vector, and determined the DNA sequence of the clones. The preliminary sequence comparison shows that C. elegans SNAP-25 is about 57% identical to the fly homolog, and 52% to the human homolog. The C. elegans SNAP-25 also shows a limited similarity to the yeast Sec9 protein (36% identity in a stretch of 50 amino acids). We screened the Barstead and Waterston cDNA library to identify full length cDNA clones. One of the cDNA clones contained 7 nucleotides identical to the 3' end of the SL-1 leader sequence, indicating that this clone contains a full length transcript. Sequencing of the clone is under way. We performed a YAC grid filter hybridization experiment using the PCR clone as a probe and identified three apparently positive YACs on chromosome V. Further physical mapping of the gene is under way.

Soares, Marcell et al. (2021) International Worm Meeting "toluene-induced bioenergetics changes generate early aging and decreased healthspan in caenorhabditis elegans"

Soares, Marcell, Baptista, Fabiane, Silveira, Tassia, Cordeiro, Larissa, Silva, Aline, Avila, Daiana, Soares, Felix

[International Worm Meeting, 2021]

aging is a normal and inevitable cellular physiological process, however, in current days, individual or environmental factors are contributors to early aging, thus generating an impaired healthspan. barker, in an ecologic psychology context mentioned that "the environment always exerts influences in the individual". if we adapt this to the toxicological sphere, daily we are exposed to many agents, whether intentional (drug's abuse) or in a non-intentional manner (environmental or occupational), that induce countless toxic effects to the organism (influence). toluene is a major solvent used in industry of paints, gasoline and adhesives, therefore, workers are the non-intentional target public. however, toluene is also a psychoactive substance and epidemiologic data have shown its abuse as the third mostly consumed in comparison to others drugs there are many studies reporting the short-term effects of toluene, nevertheless, long-term effects after end of exposure are scarce. based on that, the aim of our work was to investigate the long-term impact of airborne exposure to toluene using caenorhabditis elegans. we used two strains: n2 wild type and mutant pe255 feIs5 [sur-5p::luciferase::GFP + rol-6(su1006)] to measure atp levels. approximately 100 nematodes at l4 stage were exposed to toluene in a vapor chamber for 24h, mimetizing two scenarios of exposure: scenario 1 (mean conc. 792 ppm) and scenario 2 (mean conc. 1,094 ppm). the experiments were conducted 1, 48 and 96 hours after exposure. lipofuscin autofluorescence was measured as an aging bioindicator, neurobehaviors (head trashes, velocity and path length) to assess healthspan, and atp levels as mitochondrial function endpoint. we observed that worms exposed to both scenarios of exposure have shown increase of cellular lipofuscin accumulation, and that exposed worms demonstrated a progressive reduction of mobility in a significant manner. these findings corroborated with a decrease of mitochondrial functionality, demonstrated by atp levels reduction. here we demonstrate that toluene-induced early cellular aging can occur associate to accentuated motor loss that could be explained by a mitochondrial dysfunction, thus impacting on healthspan reduction. the literature supports this discovery, once long-term exposures either intentionally or occupationally are harmful to humans, however, here we observed that even long periods after withdrawal the toluene effects remain.