Metformin treatment of diverse Caenorhabditis species reveals the importance of genetic background in longevity and healthspan extension outcomes

Metformin, the most commonly prescribed anti‐diabetes medication, has multiple reported health benefits, including lowering the risks of cardiovascular disease and cancer, improving cognitive function with age, extending survival in diabetic patients, and, in several animal models, promoting youthful physiology and lifespan. Due to its longevity and health effects, metformin is now the focus of the first proposed clinical trial of an anti‐aging drug—the Targeting Aging with Metformin (TAME) program. Genetic variation will likely influence outcomes when studying metformin health effects in human populations. To test for metformin impact in diverse genetic backgrounds, we measured lifespan and healthspan effects of metformin treatment in three Caenorhabditis species representing genetic variability greater than that between mice and humans. We show that metformin increases median survival in three C. elegans strains, but not in C. briggsae and C. tropicalis strains. In C. briggsae, metformin either has no impact on survival or decreases lifespan. In C. tropicalis, metformin decreases median survival in a dose‐dependent manner. We show that metformin prolongs the period of youthful vigor in all C. elegans strains and in two C. briggsae strains, but that metformin has a negative impact on the locomotion of C. tropicalis strains. Our data demonstrate that metformin can be a robust promoter of healthy aging across different genetic backgrounds, but that genetic variation can determine whether metformin has positive, neutral, or negative lifespan/healthspan impact. These results underscore the importance of tailoring treatment to individuals when testing for metformin health benefits in diverse human populations.     

Species differentiation in Caenorhabditis briggsae and Caenorhabditis elegans

Identification of five laboratory strains (1-5) of putative Caenorhabditis briggsae was undertaken. Examination of the male bursal ray arrangement, mating tests with males of Caenorhabditis elegans, malate dehydrogenase zymograms, and SDS polyacrylamide electrophoresis demonstrated that strain 4 was C. briggsae and the others were C. elegans.     

The type and time of occurrence of aminopterin-induced chromosome aberrations in cultured Potorous cells

Many inhibitors of DNA synthesis have been found to induce chromosome aberrations. Our kinetic studies indicate that treatment of cellswith 10^?7M aminopterin in the presence of 10^?4M glycine, 10^?4M hypoxanthine, and 10^?4M thymidine allows continued normal cell growth. Omission of thymidine, a treatment which is known to inhibit DNA synthesis while allowing RNA and protein synthesis to continue, leads to cessation of cell growth. Treament of Potorous cell cultures with aminopterin in the presence of hypoxanthine and glycine without thymidine led to the following observations: (1) only non-exchange chromatid aberrations were formed after aminopterin treatment; (2) the aberrations were induced only in cells treated during S, and the breaks were associated with the replicating region of the chromosome; (3) breaks were observed at the first metaphase after the beginning of treatment; and (4) thymidine could reverse the chromosome-breaking action of aminopterin. A model for the molecular mechanism is suggested.     

A Genome-Wide Hybrid Incompatibility Landscape between Caenorhabditis briggsae and C. nigoni

Systematic characterization of ẖybrid incompatibility (HI) between related species remains the key to understanding speciation. The genetic basis of HI has been intensively studied in Drosophila species, but remains largely unknown in other species, including nematodes, which is mainly due to the lack of a sister species with which C. elegans can mate and produce viable progeny. The recent discovery of a C. briggsae sister species, C. nigoni, has opened up the possibility of dissecting the genetic basis of HI in nematode species. However, the paucity of dominant and visible marker prevents the efficient mapping of HI loci between the two species. To elucidate the genetic basis of speciation in nematode species, we first generated 96 chromosomally integrated GFP markers in the C. briggsae genome and mapped them into the defined locations by PCR and Next-Generation Sequencing (NGS). Aided by the marker, we backcrossed the GFP-associated C. briggsae genomic fragments into C. nigoni for at least 15 generations and produced 111 independent introgressions. The introgression fragments cover most of the C. briggsae genome. We finally dissected the patterns of HI by scoring the embryonic lethality, larval arrest, sex ratio and male sterility for each introgression line, through which we identified pervasive HI loci and produced a genome-wide landscape of HI between the two nematode species, the first of its type for any non-Drosophila species. The HI data not only provided insights into the genetic basis of speciation, but also established a framework for the possible cloning of HI loci between the two nematode species. Furthermore, the data on hybrids confirmed Haldane’s rule and suggested the presence of a large X effect in terms of fertility between the two species. Importantly, this work opens a new avenue for studying speciation genetics between nematode species and allows parallel comparison of the HI with that in Drosophila and other species.     

Étude de l'action de l'aminoptérine sur la recombinaison génique chez Drosophila melanogaster

Study of the action of aminopterin on genic recombination in Drosophila melanogaster—A study was made of the action, during meiosis, of aminopterin, inhibitor of nucleotide metabolism. An increase of the rate of crossing over was observed. This increase was studied on two systems, by using highly linked markers, so as to minimize the amount of double crossing over: vg-su, on chromosome II and y-w ^a, on the X chromosome. It is shown that aminopterin induces a significant increase of recombination. The independence of the two systems makes plausible that aminopterin could act on the whole genome at a meiotic level.     

The high-level accumulation of n-3 polyunsaturated fatty acids in transgenic pigs harboring the n-3 fatty acid desaturase gene from Caenorhabditis briggsae

Livestock meat is generally low in n-3 polyunsaturated fatty acids (PUFAs), which are beneficial to human health. An alternative approach to increasing the levels of n-3 PUFAs in meat is to generate transgenic livestock animals. In this study, we describe the generation of cloned pigs that express the cbr-fat-1 gene from Caenorhabditis briggsae, encoding an n-3 fatty acid desaturase. Analysis of fatty acids demonstrated that the cbr-fat-1 transgenic pigs produced high levels of n-3 fatty acids from n-6 analogs; consequently, a significantly reduced ratio of n-6/n-3 fatty acids was observed. We demonstrated that the n-3 desaturase gene from C. briggsae was functionally expressed, and had a significant effect on the fatty acid composition of the transgenic pigs, which may allow the production of pork enriched in n-3 PUFAs.     

AP endonuclease deficiency results in extreme sensitivity to thymidine deprivation

Thymidine depletion is toxic to virtually all actively growing cells. The fundamental mechanism responsible for thymidineless death remains unknown. One event thought to be critical in causing the toxicity of thymidine depletion is a sharp rise in the ratio of dUTP to dTTP and subsequent incorporation of dUTP into DNA. Maneuvers to alter dUTP levels appear to alter the toxicity of thymidine depletion. However, loss of uracil-DNA-N-glycosylase activity does not appear to change the toxicity of thymidine deprivation significantly. This study proposes to define the role of uracil base excision repair (BER) in mediating thymidineless death. The toxicity of thymidine deprivation induced by the antifolate aminopterin was measured in a series of mutant Saccharomyces cerevisiae strains deficient in various steps in uracil-BER. Most mutants displayed modest changes in their sensitivity to aminopterin, with the exception of cells lacking the abasic endonuclease Apn1. apn1 mutants displayed a profound sensitivity to aminopterin that was relieved in an apn1 ung1 double mutant. Wild-type and apn1 mutants displayed similar levels of DNA damage and S-phase arrest during aminopterin treatment. A significant portion of cell killing occurred after removal of aminopterin in both wild-type and apn1 mutant cells. apn1 mutants showed a complete inability to re-initiate DNA replication following removal of aminopterin. These findings suggest recovery from arrest is a crucial step in determining the response to thymidine deprivation and that interruptions in uracil-BER increase the toxicity of thymidine deprivation by blocking re-initiation of replication rather than inciting global DNA damage. Inhibition of apurinic/apyrimidinic endonuclease may therefore be a reasonable approach to increase the efficacy of anticancer chemotherapies based on thymidine depletion.     

Coevolution within and between Regulatory Loci Can Preserve Promoter Function Despite Evolutionary Rate Acceleration

Phenotypes that appear to be conserved could be maintained not only by strong purifying selection on the underlying genetic systems, but also by stabilizing selection acting via compensatory mutations with balanced effects. Such coevolution has been invoked to explain experimental results, but has rarely been the focus of study. Conserved expression driven by the unc-47 promoters of Caenorhabditis elegans and C. briggsae persists despite divergence within a cis-regulatory element and between this element and the trans-regulatory environment. Compensatory changes in cis and trans are revealed when these promoters are used to drive expression in the other species. Functional changes in the C. briggsae promoter, which has experienced accelerated sequence evolution, did not lead to alteration of gene expression in its endogenous environment. Coevolution among promoter elements suggests that complex epistatic interactions within cis-regulatory elements may facilitate their divergence. Our results offer a detailed picture of regulatory evolution in which subtle, lineage-specific, and compensatory modifications of interacting cis and trans regulators together maintain conserved gene expression patterns.     

The vestigial locus of Drosophila melanogaster is involved in resistance to inhibitors of dTMP synthesis

The vestigal (vg) gene encodes a nuclear protein which plays a major role in the formation of the wing of Drosophila. Resistance or sensitivity to aminopterin, an inhibitor of the dihydrofolate reductase enzyme in D. melanogaster, seems to be associated with a specific alteration in vg gene function. Wild-type and vg mutant strains selected for growth on increasing concentrations of aminopterin display changes in physiological and biochemical parameters such as viability on normal and aminopterin-containing media, duration of development, wing phenotype, dihydrofolate reductase activity, and cross-resistance to fluorodeoxyuridine (FUdR) and to methotrexate. Our results indicate that the mechanisms of resistance differ in the wild-type and mutant strains. The vg ^83b27 mutant, in which the major part of intron 2 of the vg gene is deleted, is associated with a high rate of resistance to FUdR, an inhibitor of thymidylate synthetase. Moreover, vg ^83b27/vg ^BGheterozygotes, which are wild type when grown on normal medium, display a strong vg phenotype when grown on aminopterin. Our results indicate a role for the vestigial locus in mediating resistance to inhibitors of dTMP synthesis.     

Nematode selenoproteome: the use of the selenocysteine insertion system to decode one codon in an animal genome?

Selenocysteine (Sec) is co-translationally inserted into selenoproteins in response to codon UGA with the help of the selenocysteine insertion sequence (SECIS) element. The number of selenoproteins in animals varies, with humans having 25 and mice having 24 selenoproteins. To date, however, only one selenoprotein, thioredoxin reductase, has been detected in Caenorhabditis elegans, and this enzyme contains only one Sec. Here, we characterize the selenoproteomes of C.elegans and Caenorhabditis briggsae with three independent algorithms, one searching for pairs of homologous nematode SECIS elements, another searching for Cys- or Sec-containing homologs of potential nematode selenoprotein genes and the third identifying Sec-containing homologs of annotated nematode proteins. These methods suggest that thioredoxin reductase is the only Sec-containing protein in the C.elegans and C.briggsae genomes. In contrast, we identified additional selenoproteins in other nematodes. Assuming that Sec insertion mechanisms are conserved between nematodes and other eukaryotes, the data suggest that nematode selenoproteomes were reduced during evolution, and that in an extreme reduction case Sec insertion systems probably decode only a single UGA codon in C.elegans and C.briggsae genomes. In addition, all detected genes had a rare form of SECIS element containing a guanosine in place of a conserved adenosine present in most other SECIS structures, suggesting that in organisms with small selenoproteomes SECIS elements may change rapidly.     

Plasmodium falciparum: in vitro induction of resistance to aminopterin

Plasmodium falciparum parasites were grown on microplates in the presence of aminopterin. The FCR-8 strain was more sensitive to aminopterin than a Richards strain and died within 1 week of treatment. A few parasites of the Richards strain survived treatment and developed normal parasitemias. This strain was resistant to aminopterin at concentrations not higher than those used for its selection. Removal of aminopterin did not affect the growth of the resistant variant, showing that it was not aminopterin dependent. Aminopterin affected the sensitive parasites by interfering with nucleic acid synthesis, whereas protein synthesis was not impaired. Gametocytogenesis was unaffected by aminopterin.     

Caenorhabditis briggsae: aging and the structural turnover of the outer cuticle surface and the intestine.

The exposed surface of Caenorhabditis briggsae was examined for the presence of neuraminic acid, hyaluronic acid, and glucuronic acid. None of these molecules was detected. In young nematodes the presence of a surface coat was demonstrated. This surface coat appeared to shrink with age. Ruthenium red staining suggested the presence of acid mucopoly-saccharides on the outer surface. Feeding the nematodes on cationized ferritin enabled visualization of a matrix surrounding the intestinal brush border. Experiments with an inhibitor of acid mucopolysaccharide synthesis suggested that there is no turnover of acid mucopolysaccharides after the final molt of C. briggsae.     

Comparative analysis of embryonic cell lineage between Caenorhabditis briggsae and Caenorhabditis elegans

Comparative genomic analysis of important signaling pathways in Caenorhabditis briggsae and Caenorhabditis elegans reveals both conserved features and also differences. To build a framework to address the significance of these features we determined the C. briggsae embryonic cell lineage, using the tools StarryNite and AceTree. We traced both cell divisions and cell positions for all cells through all but the last round of cell division and for selected cells through the final round. We found the lineage to be remarkably similar to that of C. elegans. Not only did the founder cells give rise to similar numbers of progeny, the relative cell division timing and positions were largely maintained. These lineage similarities appear to give rise to similar cell fates as judged both by the positions of lineally equivalent cells and by the patterns of cell deaths in both species. However, some reproducible differences were seen, e.g., the P4 cell cycle length is more than 40% longer in C. briggsae than that in C. elegans (p < 0.01). The extensive conservation of embryonic development between such divergent species suggests that substantial evolutionary distance between these two species has not altered these early developmental cellular events, although the developmental defects of transpecies hybrids suggest that the details of the underlying molecular pathways have diverged sufficiently so as to not be interchangeable.     

New Tools for Investigating the Comparative Biology of Caenorhabditis briggsae and C. elegans

Comparative studies of Caenorhabditis briggsae and C. elegans have provided insights into gene function and developmental control in both organisms. C. elegans is a well developed model organism with a variety of molecular and genetic tools to study gene functions. In contrast, there are only very limited tools available for its closest relative, C. briggsae. To take advantage of the full potential of this comparative approach, we have developed several genetic and molecular tools to facilitate functional analysis in C. briggsae. First, we designed and implemented an SNP-based oligonucleotide microarray for rapid mapping of genetic mutants in C. briggsae. Second, we generated a mutagenized frozen library to permit the isolation of targeted deletions and used the library to recover a deletion mutant of cbr-unc-119 for use as a transgenic marker. Third, we used the cbr-unc-119 mutant in ballistic transformation and generated fluorescently labeled strains that allow automated lineaging and cellular resolution expression analysis. Finally, we demonstrated the potential of automated lineaging by profiling expression of egl-5, hlh-1, and pha-4 at cellular resolution and by detailed phenotyping of the perturbations on the Wnt signaling pathway. These additions to the experimental toolkit for C. briggsae should greatly increase its utility in comparative studies with C. elegans. With the emerging sequence of nematode species more closely related to C. briggsae, these tools may open novel avenues of experimentation in C. briggsae itself.     

Natural variation in life history and aging phenotypes is associated with mitochondrial DNA deletion frequency in Caenorhabditis briggsae

Background  

Mutations that impair mitochondrial functioning are associated with a variety of metabolic and age-related disorders. A barrier to rigorous tests of 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 system, we studied natural variation in life history, metabolic, and aging phenotypes as it relates to levels of a naturally-occurring heteroplasmic mitochondrial ND5 deletion recently discovered to segregate among wild populations of the soil nematode, Caenorhabditis briggsae. The normal product of ND5 is a central component of the mitochondrial electron transport chain and integral to cellular energy metabolism.     

Design and regioselective synthesis of a new generation of targeted therapeutics. Part 3: Folate conjugates of aminopterin hydrazide for the treatment of inflammation

Efficient syntheses of folate receptor (FR) targeting conjugates of the anti-inflammatory, aminopterin hydrazide, are described. 2-{4-Benzoylamino}-5-oxo-5-{N′-[2-(pyridin-2-yldisulfanyl)-ethoxycarbonyl]-hydrazino}-pentanoic acid is synthesized from commercially available 4-[(2-amino-4-imino-3,4-dihydro-pteridin-6-yl-methyl)-amino]-benzoic acid. Conjugation of this novel, activated aminopterin hydrazide to folic acid through cysteine-terminating (C-terminus), peptide/carbohydrate spacers results in highly water soluble conjugates which allow for the release of free aminopterin hydrazide within the endosomes of targeted cells.     

Comparison of C. elegans and C. briggsae genome sequences reveals extensive conservation of chromosome organization and synteny

To determine whether the distinctive features of Caenorhabditis elegans chromosomal organization are shared with the C. briggsae genome, we constructed a single nucleotide polymorphism–based genetic map to order and orient the whole genome shotgun assembly along the six C. briggsae chromosomes. Although these species are of the same genus, their most recent common ancestor existed 80–110 million years ago, and thus they are more evolutionarily distant than, for example, human and mouse. We found that, like C. elegans chromosomes, C. briggsae chromosomes exhibit high levels of recombination on the arms along with higher repeat density, a higher fraction of intronic sequence, and a lower fraction of exonic sequence compared with chromosome centers. Despite extensive intrachromosomal rearrangements, 1:1 orthologs tend to remain in the same region of the chromosome, and colinear blocks of orthologs tend to be longer in chromosome centers compared with arms. More strikingly, the two species show an almost complete conservation of synteny, with 1:1 orthologs present on a single chromosome in one species also found on a single chromosome in the other. The conservation of both chromosomal organization and synteny between these two distantly related species suggests roles for chromosome organization in the fitness of an organism that are only poorly understood presently.     

Autosomal genes of autosomal/X-linked duplicated gene pairs and germ-line proliferation in Caenorhabditis elegans

We report molecular genetic studies of three genes involved in early germ-line proliferation in Caenorhabditis elegans that lend unexpected insight into a germ-line/soma functional separation of autosomal/X-linked duplicated gene pairs. In a genetic screen for germ-line proliferation-defective mutants, we identified mutations in rpl-11.1 (L11 protein of the large ribosomal subunit), pab-1 [a poly(A)-binding protein], and glp-3/eft-3 (an elongation factor 1-α homolog). All three are members of autosome/X gene pairs. Consistent with a germ-line-restricted function of rpl-11.1 and pab-1, mutations in these genes extend life span and cause gigantism. We further examined the RNAi phenotypes of the three sets of rpl genes (rpl-11, rpl-24, and rpl-25) and found that for the two rpl genes with autosomal/X-linked pairs (rpl-11 and rpl-25), zygotic germ-line function is carried by the autosomal copy. Available RNAi results for highly conserved autosomal/X-linked gene pairs suggest that other duplicated genes may follow a similar trend. The three rpl and the pab-1/2 duplications predate the divergence between C. elegans and C. briggsae, while the eft-3/4 duplication appears to have occurred in the lineage to C. elegans after it diverged from C. briggsae. The duplicated C. briggsae orthologs of the three C. elegans autosomal/X-linked gene pairs also display functional differences between paralogs. We present hypotheses for evolutionary mechanisms that may underlie germ-line/soma subfunctionalization of duplicated genes, taking into account the role of X chromosome silencing in the germ line and analogous mammalian phenomena.     

Enigma variations: control of sexual fate in nematode germ cells

A new study showing that neither FEM-2 nor FEM-3 is required for spermatogenesis in Caenorhabditis briggsae, unlike in Caenorhabditis elegans, implies that the sex-determination pathway in these species is evolving rapidly, and supports the proposal that they evolved hermaphroditism independently.