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The Caenorhabditis elegans SLC database: Solute transporter identification using comparative genomics

Carsten Jäckel1, Hannelore Daniel1 and Britta Spanier1
1Molecular Nutrition Unit, Technical University of Munich, Freising, Germany

Correspondence to: Britta Spanier (spanier@wzw.tum.de)

Comparative genomic approaches have proven to be powerful tools in identifying genes as well as categorizing them based on protein functions. One product is the HGNC (Human Gene Nomenclature Committee) Solute Carrier (SLC) System (Hediger et al., 2004; He et al., 2009). It comprises 55 families with a total of over 365 putative proteins that act as uniporters, symporters or antiporters in the plasma cell membrane as well as in mitochondrial and vesicular membranes. The range of transported substrates covers inorganic and organic cations/anions, amino acids and peptides, sugars, bile salts, urea, biogenic amines, neurotransmitters, ammonium, choline, vitamins, nucleosides/nucleotides, fatty acids, etc. In addition, numerous orphan transporters are contained that need their substrates be defined. Evidently these transporters are of critical importance for cellular homeostasis and thus many of these genes are highly conserved across species. Here we present the C. elegans SLCdb (www.wormslc.org), a database that lists the top C. elegans homologs to the mammalian SLC tables. This database allows for a quick identification of target genes by either directly searching for worm or SLC gene names or aliases and for retrieval of available information. The site also features a quick overview of all SLC families and the currently published knowledge on these transporters in C. eleganswith a reference list.

To date, the database contains 1886 homology datasets. The homologous C. elegans proteins have been selected step-wise. First the top BLASTP (Altschul et al., 1990) hits were selected, applying a cutoff threshold of E-20, which removes most false positives. In a next step genes that have been published but may have been below the threshold were manually added to the list. Since BLAST algorithms only do local alignments they do not account for protein lengths and overall homology. Because of that limitation we next used the NEEDLE algorithm (Needleman et al., 1970) for additional information and confirmation for the results obtained. For assessing of how well conserved a protein is within the nematode phylum, every single protein was then compared to its closest C. briggsae homolog (using NEEDLE), which usually yielded very high sequence identities and similarities. Finally, all results were compiled into a uniform format in the database, presenting the results sorted by NEEDLE score, showing the closest C. elegans homologs, a brief description of the protein functions (when available), links to WormBase and the C. briggsae homolog along with a brief description and the NEEDLE score.

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Figure 1:

References

Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. (1990). Basic local alignment search tool. J. Mol. Biol. 215, 403-410. PubMed

He L, Vasiliou K, Nebert DW. (2009). Analysis and update of the human solute carrier (SLC) gene superfamily. Hum. Genomics 3, 195-206. PubMed

Hediger MA, Romero MF, Peng JB, Rolfs A, Takanaga H, Bruford EA. (2004). The ABCs of solute carriers: physiological, pathological and therapeutic implications of human membrane transport proteins. Pflugers Arch. 447, 465-468. PubMed

Needleman SB, Wunsch CD. (1970). A general method applicable to the search for similarities in the amino acid sequence of two proteins. J. Mol. Biol. 48, 443-453. PubMed

Naming genes beyond Caenorhabditis

Robin N. Beech1, Dante Zarlenga3, David Bird4, Matt Berriman5 and Joe Dent2
1Parasitology, McGill University, Montreal, QC, Canada, 2Biology, McGill University, Montreal, QC, Canada, 3Animal Parasitic Diseases, USDA, Beltsville MD, 4Plant Nematode Genomes Group, NC State University, Raleigh NC, 5Wellcome Trust Sanger Institute, Cambridge, UK

Correspondence to: Robin N. Beech (robin.beech@mcgill.ca)

The nomenclature of genes in Caenorhabditis elegans is based on long-standing, successful guidelines established in the late 1970s (Horvitz et al., 1979). Over time these guidelines have matured into a comprehensive, systematic nomenclature that is easy to apply, descriptive and therefore highly informative.

Recently, a flood of parasitic nematodes genome data has become available, including Brugia (Ghedin et al., 2007) and Megliodogyne (Opperman et al., 2008). Annotation of these genomes is largely based on blast search similarity with C. elegans as the closest and most thoroughly characterized nematode. Several different nomenclature systems have developed, as seemed appropriate to those involved in each genome project. A strong case can be made for the general adoption of the C. elegansnomenclature guidelines across the nematodes (Beech et al., 2010; Bird and Riddle, 1994), although until recently this has not been widely used.

A two, and later, three letter species code allowed the C. elegans nomenclature to be extended to four additional Caenorhabditis species C. japonicum (Cja), C. ramanii (Cra), C. brigssae (Cbr) and C. brenneri (Cbe). Applying the guidelines to a wide range of nematode species will require further modification. Using a four letter species code could identify 1364 of the 1537 nematode species listed with NCBI, without modification, rather than 976. C. elegans genes with several paralogs in a related species can be identified with a period and number after the gene name. For example, four copies of unc-29 in the trichostrongylid parasites are unc-29.1, unc-29.2, unc-29.3 and unc-29.4 (Neveu et al., 2010). With no defined wildtype for parasitic species, a standard for designating polymorphism needs to be developed. Finally, among the 50-60% of genes in distantly related nematodes that have no clearly identifiable C. elegans homolog, are likely to be those that are most interesting for organism biology. Naming of these species-specific genes requires coordination with the C. elegans nomenclature to avoid conflicts. For a nomenclature to be accepted and generally applied, a wide consensus among those who use the guidelines is required. A workshop will be held at the ICOPA XII conference in Melbourne, August 2010, to make recommendations for new guidelines.

References

Beech RN, Wolstenholme AJ, Neveu C, Dent JA. (2010). Nematode parasite genes, what’s in a name? Trends Parasitol. [Epub ahead of print]  PubMed

Bird DM, Riddle DL. (1994). A genetic nomenclature for parasitic nematodes. J. Nematol. 26, 138-143.  PubMed

Ghedin E, Wang S, Spiro D, Caler E, Zhao Q, Crabtree J, Allen JE, Delcher AL, Guiliano DB, Miranda-Saavedra D, et al. (2007). Draft genome of the filarial nematode parasite Brugia malayi. Science 317, 1756-1760.  PubMed

Horvitz HR, Brenner S, Hodgkin J, Herman RK. (1979). A uniform genetic nomenclature for the nematode Caenorhabditis elegans. Mol. Gen. Genet. 175, 129-133.  PubMed

Neveu C, Charvet C, Fauvin A, Cortet J, Beech R, Cabaret, J. (2010). Genetic diversity of levamisole receptor subunits in parasitic nematodes and abbreviated transcripts associated with resistance. Pharmacogenetics and Genomics In Press.  PubMed

ENGM: an NGM-like solid media suitable for doing genetics on the entomopathogenic nematode Heterorhabditis bacteriophora

András Fodor1,2, Andrea Máthé-Fodor1,2, Éva Lehoczky1, Ganpat Jagdale2, Parwinder S. Grewal2 and Michael G. Klein2
1Institute of Plant Protection, Georgikon Faculty, University of Pannonia, Keszthely, Hungary, 2Department of Entomology, Ohio State University, OARDC, Wooster OH

Correspondence to: András Fodor (fodorandras@yahoo.com)

There are two “secret weapons” behind the scientific success of C. elegans: NGM media and OP50 bacteria. The Heterorhabditis bacteriophora/Photorhabdus luminescens entomopathogenic nematode/bacterium (EPN/EPB) complexes are widely used in biological control. Therefore elaboration of genetic toolkits for EPN species would have a practical impact. C. elegans is considered as a model for genetic analysis of EPN species (Fodor et al., 1990). The genome sequence of H. bacteriophora (H. b.) is almost complete. The “secret tools” (the proper NGM-like media and OP50-like bacteria) for genetics of H. b. have, however, been missing. H. b. can only be fed on its own symbiont in lab media, which is rich in N sources and contains oil. Neither H. b. nor its bacterial symbiont could grow in NGM, even if it was supplemented with oil and an abundant peptone N-source. The results were hardly better with supplemented TSY or TSA. In a rich oily media (Woots, NA) the bacteria overgrow the nematodes and the animals cannot be observed individually. In Woots agar media the growth was remarkable, but the visibility of the nematodes was poor. In Woots agar C. elegans could propagate, but “feeding RNAi” (Fire et al., 1998) could not be induced. We elaborated a novel media, called ENGM (Entomopathogenic Nematode Growth Media) in which H. b., C. elegans, as well as their food-source bacteria (P. luminescens, E. coli TT01, OP50) could properly grow. The visibility of the nematodes on ENGM is almost as good as that on NGM. Feeding RNAi can be induced in C. elegansby feeding in both NGM and ENGM with similar frequencies, suggesting that RNAi in H. b. might also be studied in ENGM. Since then we successfully induced heterolog RNAi in H. b. (in preparation). The food source for H. b. is the moderately growing Tn-10-induced NS107 rifR, kmR, apR mutant (isolated by us) of P. luminescens TT01 (Duchaud E. et al., 2003) (kindly provided by Dr. T. Ciche). The recipe of ENGM is as follows: 2.5 g bacto-peptone; 1.5 g beef extract; 2.3 g brain-heart infusion; 15 g agar to 1 liter of deionized water. After autoclaving: 5 g vegetable oil; 1 ml of 5 mg/ml cholesterol (dissolved in EtOH); 2 ml of 0.5M MgSO4, rifampicin100, kanamycin30) are to be added. ENGM plates should be seeded with moderately growing symbiotic bacteria, such as NS107. (For details on the isolation of NS107, please contact the corresponding author).

References

Duchaud E, Rusniok C, Frangeul L, Buchrieser C, Givaudan A, Taourit S, Bocs S, Boursaux-Eude C, Chandler M, Charles JF, et al. (2003). The genome sequence of the entomopathogenic bacterium Photorhabdus luminescens Nat. Biotechnol. 21, 1307 – 1313.  PubMed

Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC. (1998). Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 19, 806-811.  PubMed

Fodor A, Vecseri G, Farkas T. (1990). C. elegans, as a model for studying entomopathogenic nematodes. In Entomopathogenic Nematodes in Biological Control, R. Gaugler and H. Kaya eds. (Boca Raton, FL, USA: CRC Press), pp. 285-300.  PubMed

Potential for cytonuclear epistasis in Caenorhabditis briggsae inter-population hybrids

Anna L. Coleman-Hulbert1, Dana K. Howe2, Dee R. Denver2 and Suzanne Estes1
1Department of Biology, Portland State University, Portland OR, 2Department of Zoology and Center for Genome Research and Biocomputing, Oregon State University, Corvallis OR

Correspondence to: Suzanne Estes (estess@pdx.edu)

A barrier to rigorous tests for the role of mitochondrial dysfunction in aging processes has been the lack of model systems with relevant, naturally occurring mitochondrial genetic variation. Toward the goal of developing such a model, we studied life history, metabolic, and aging phenotypes in natural and experimental populations of Caenorhabditis briggsae harboring different levels of a heteroplasmic mitochondrial ND5 deletion recently discovered to segregate among many wild C. briggsae populations (Howe and Denver, 2008). The normal product of ND5 is a central component of complex I of the mitochondrial electron transport chain and integral to cellular energy metabolism.

Natural high-ND5 deletion C. briggsae isolates have decreased total fecundity (Howe and Denver, 2008). We found that heteroplasmic C. briggsae also have reduced pharyngeal pumping rates throughout adulthood and grow faster and larger as larvae, but slower as adults, compared to zero-deletion isolates. Hybrid strains were constructed by reciprocally crossing pairs of high- and low-heteroplasmy populations in order to isolate the mitochondrial genome of one population onto the nuclear genetic background of another population. F10 individuals were assayed for the above traits with the expectation that hybrid means would match those of the mitochondrial source population if ND5 deletion heteroplasmy level is responsible for the phenotypic variation among C. briggsae isolates. However, C. briggsae hybrid means often differed from those of maternal lineage and from each other for all traits (e.g., Fig. 1). Because coordination between nuclear and mitochondrial gene products is required for optimal energy metabolism, our results may indicate cytonuclear conflict and heterosis in hybrid C. briggsae, but paternal transmission of mitochondria has yet to be eliminated as a potential cause of these results.

Figures



Figure 1: Lifetime fecundity in high- (HK105) and low-ND5 deletion (PB800) C. briggase isolates and reciprocal hybrids. mHnP_1-3 are hybrid replicates in which HK105 was the mitochondrial donor and PB800 was the nuclear donor. The reverse is true for mPnH_1-3. Lines = medians, boxes encompass 50% of data, vertical lines encompass 10th-90th percentiles, circles = individual outliers.

References

Howe DK, and Denver DR. (2008). Muller’s ratchet and compensatory mutation in Caenorhabditis briggsae mitochondrial genome evolution. BMC Evol. Biol. 8, 62. PubMed

Comparison of Caenorhabditis elegans and Caenorhabditis briggsae for thermotolerance features

Marat Kh. Gainutdinov1, Alya Kh. Timoshenko1, Rufina R. Kolsanova1, Timur M. Gainutdinov1 and Tatyana B. Kalinnikova1
1Research Institute for Problems of Ecology and Mineral Wealth Use of Tatarstan Academy of Sciences, Kazan, Russian Federation

Correspondence to: Marat Kh. Gainutdinov (mgainutdinov@nm.ru)

Thermotolerance evolution of poikilothermic organisms consists in adaptation to temperature conditions of habitats of such physiological features as the lower and upper limits of temperature for reproduction, development and behavior and organism’s resistance to short-term temperature extremes.

Previous researches of Caenorhabditis revealed both interspecific and intraspecific significant differences for the upper limits of temperature for reproduction and development. These differences are in accordance with temperature conditions of habitats (Fatt and Dougherty, 1963; Fodor et al., 1983). Nevertheless interspecific differences in tolerance to temperature extremes are still uninvestigated.

Therefore we compared resistance to high temperature extremes of C. elegans (strain N2) and C. briggsae (strain AF16). Experiments were carried out with young adults grown in Petri dishes with NGM and E.coli OP50 at 23°C. Resistance to constant temperature 36°C or 37°C was measured for worms incubated individually in 1 ml of liquid medium (NGM without agar, peptone, cholesterol).

Results of our experiments show that organism’s resistance to extreme high temperature is significantly higher in C. briggsae than in C. elegans:

  1. Mean time of reversible disturbances of swimming induced by intensive mechanic stimulus (shaking of test tube) was similar at 36°C in C. elegans and at 37°C in C. briggsae.
  2. Mean time of heat death was rather more in C. briggsae at 37°C than in C. elegans at 36°C.

Rapid adaptations of worms to temperature elevation by 2 hour incubation at 30°C or by heat hardening (1 hour at 33°C followed by 1 hour at 23°C) increased behavior resistance to short-term heat stress (36°C for C. elegans and 37°C for C. briggsae). Efficiency of both adaptations was similar in C. elegans and C. briggsae.

Therefore we conclude that adaptation of C. elegans and C. briggsae to different temperature conditions of habitats changed only base resistance to extreme high temperature.

It is known that the upper temperature limit for reproduction of C. elegans N2 is 26.5°C (Fatt and Dougherty, 1963), while C. briggsae AF16 can grow at 27.5°C (Gupta et al., 2007). Therefore our data indicate that evolution of Caenorhabditis thermotolerance involves unidirectional adaptive changes of the upper limits of temperature for reproduction and resistance to short-term heat stress. This pattern reveals in the evolution of most poikilotherms.

Thus soil nematodes Caenorhabditis may be used as convenient model organisms for study thermotolerance evolution.

References

Fatt HV, Dougherty EC. (1963). Genetic control of differential heat tolerance in two strains of the nematode, Caenorhabditis elegans. Science. 141, 266-267.  PubMed

Fodor A, Riddle DL, Nelson FK, Golden JW. (1983). Comparison of a new wild-type Caenorhabditis briggsae with laboratory strains of C. briggsae and C. elegans. Nematologica 29, 203–217. Abstract  PubMed

Gupta BP, Johnsen R, Chen N. (2007). Genomics and biology of the nematode Caenorhabditis briggsae, Wormbook, ed. The C. elegans Research Community, Wormbook, doi/10.1895/wormbook.1.136.1, http//www.wormbook.org.  PubMed

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