Phillips, Carolyn M. et al. (2011) International Worm Meeting "Characterization of Transposon Silencing Pathways."

Ruvkun, Gary B., Phillips, Carolyn M.

[International Worm Meeting, 2011]

Small RNA mediated silencing of transposons is an important mechanism to prevent DNA damage and subsequent mutations in the germline. A subset of the exogenous and endogenous small RNA pathways genes have been demonstrated to be involved in transposon silencing. When these genes are mutated, transposons can be excised, leaving a double-strand break, which must be repaired by the cellular machinery, either through homologous recombination or non-homologous end joining. I have developed a strategy to visualize double-strand breaks in the germline generated by unregulated transposons in the transposon silencing mutants. This assay will allow me to look at transposon hopping in mutants that are sterile and possibly to perform a small-scale cytological screen for new genes in the transposon silencing pathway. Additionally, I am generating fluorescently tagged proteins to perform localization analysis of both new and previously characterized genes in this pathway. Localization may identify particular cells where RNA based immunity is important or sub-cellular structures to which these proteins concentrate. Finally, to identify direct binding partners of these proteins and potentially identify additional components of the transposon silencing machinery, I will biochemically purify the components of this pathway. Interacting proteins will be identified by mass spectrometry. Any components identified through this strategy can be further characterized by mutational and localization analysis. These experiments will examine the relationship between the small RNA pathways and genome integrity, and further elucidate the mechanisms of transposon silencing in the germline. CMP is the Marion Abbe Fellow of the Damon Runyon Cancer Research Foundation.

Phillips, Carolyn M. et al. (2009) International Worm Meeting "Characterization of the Transposon Silencing Machinery."

Phillips, Carolyn M., Ruvkun, Gary

[International Worm Meeting, 2009]

Small RNA-mediated gene silencing is a conserved mechanism used by nearly all eukaryotes as a way to regulate gene expression at both the transcriptional and post-transcriptional level. The double-strand RNA can be delivered from outside the cell or can be generated from endogenous transcription of coding and non-coding genomic loci. Some processes known to be regulated by RNAi include transposon silencing, defense against viral infection, and maintenance of heterochromatin. Transposon silencing is an important mechanism to prevent DNA damage and subsequent mutations in the germline. A subset of the genes shown to be involved in exogenous RNAi have also been shown to be involved in transposon silencing in the germline, including mut-2, mut-7, mut-14, mut-15, mut-16, and rde-2. When these genes are mutated, transposons can be excised, leaving a double-strand break (DSBs), which must be repaired by the cellular machinery, either through homologous recombination or non-homologous end joining. I am currently developing a strategy to visualize DSBs in the germline generated by unregulated transposons in the transposon silencing mutants. This assay will allow me to perform a small-scale cytological screen for new genes in the transposon silencing pathway. Additionally, I am generating fluorescently tagged proteins to perform localization analysis of both new and previously characterized genes in this pathway. Localization may identify particular cells where RNA based immunity is important or sub-cellular structures to which these proteins concentrate. Finally, to identify direct binding partners of these proteins and potentially identify additional components of the transposon silencing machinery, I will biochemically purify the components of this pathway. The tagged constructs that I am generating for cytology will include a modified version of the Tandem Affinity Purification (TAP) tag generated by Cheesman et al. (2004), which includes a fluorescent tag followed by a TEV protease cleavage site and the S peptide domain and is referred to as the Localization and Affinity Purification (LAP) tag. It allows for both visualization of the protein and its affinity purification. Interacting proteins will be identified by mass spectrometry. Any components identified through this strategy can be further characterized by mutational and localization analysis. These experiments will examine the relationship between the RNAi pathway and the maintenance of genome integrity, and further elucidate the mechanisms of transposon silencing in the germline.

Phillips PC (1995) International C. elegans Meeting "DISTINGUISHING CHAOS FROM NOISE IN NEMATODE POPULATION DYNAMICS"

Phillips PC

[International C. elegans Meeting, 1995]

C. elegans populations raised via serial transfer with discrete generations display fluctuations in population size that can span several orders of magnitude. This is somewhat unusual since the worms are maintained under controlled conditions on a fixed amount of resource (OP50). Large fluctuations in population size in an apparently constant environment could be the signature of deterministic chaos induced by density dependent population size regulation and the very large reproductive capacity of C. elegans. To test this hypothesis, I raised four replicate lines of worms for 100 generations at 20 C, transferring the populations every four days using the hypochlorite cleaning procedure (i.e., only eggs were transferred). Analysis of the resulting time series shows that there is strong density dependent regulation in these populations, but that the size fluctuations are driven mostly by environmental variation rather than deterministic chaos. The environmental influences are probably caused by variation in the age and quality of E. coli cultures and plates, as well as small differences in the timing of transfers. Large scale sensitivity resulting from small amounts of input variation probably arises from "local chaos" during different parts of the population regulation cycle. This study demonstrates that C. elegans can be developed into a useful model system for ecological and evolutionary studies.

Phillips, Patrick et al. (2015) International Worm Meeting "Caenorhabditis Intervention Testing Program: Gateway to the Fountain of Youth."

Guo, Max, Lithgow, Gordon, Driscoll, Monica, Phillips, Patrick, Harke, Jailynn

[International Worm Meeting, 2015]

The Caenorhabditis Intervention Testing Program (CITP) is a multi-institution effort, sponsored by the National Institute of Aging, designed to screen bioactive compounds for their ability to extend lifespan and enhance health using nematodes as a model system for the potential effects of background genetic variation. The Intervention Testing Program (ITP), began assaying pharmacological interventions for lifespan extension in mice in the early 2000s [1]. However, with a median lifespan over two years, this project has decreased capacity to test multiple compounds in a robust manner [2]. The CITP capitalizes on the short lifespan of hermaphroditic C. elegans, C. briggsae and C. tropicalis. Selected natural isolates represent a biologically and geographically diverse population, yielding a total of 22 test strains. A compound effective across a diverse worm panel increases the likelihood of potency across other species, including heterogeneous human populations.Reproducibility marks a key goal of the CITP; the collaborative group generated detailed standardized protocols to minimize among and within lab variability. A data center facilitates near-instantaneous and global access to all CITP data. Compound candidates are selected based on the scientific community's recommendations and advice from the project's steering committee.A subset of strains has already undergone initial testing of four compound interventions: (R) alpha-lipoic acid, Aspirin, Quercetin, and NP1. Resveratrol, propyl gallate, and Thioflavine T are to be tested shortly. In addition to manual lifespan experiments, each lab built over 20 automated lifespan machines which use flatbed scanner technology [3], allowing for high throughput and precise longevity measurements. Mid- and late-age health assays are also underway.Initial results indicate significant differences in among-isolate variation in longevity within species (e.g., C. elegans low variation, C. briggsae high variation), as well as strain- and species-specific differences in longevity responses to compound treatments.1. Warner et al (2000) Mech Ageing Dev 115:199-2072. Miller et al (2007) Aging Cell 6:565-5753. Stroustrup et al (2013) Nature Methods, 10:665-670.

Phillips, Barret C. et al. (2009) International Worm Meeting "Forward Genetic Analysis of the Synaptic Gas Pathway."

Phillips, Barret C., Edwards, Stacey L., Miller, Kenneth G.

[International Worm Meeting, 2009]

Neurons communicate with each other and with muscle cells at synapses. A long term goal of this lab is to bridge the gap between synaptic function and the control of behavior by determining how signal transduction pathways regulate the activity of synapses. Our studies focus on the neuromuscular synapses that control locomotion in C. elegans. Genetic studies have shown that the integrated activities of at least 4 major, conserved Ga pathways control synaptic activity to produce the C. elegans locomotion behavior. Within this network, the neuronal Gas pathway is an especially critical, but poorly understood, link between synaptic function and behavior- driving both primordial synaptic functions, such as C. elegans locomotion, and higher synaptic functions such as sleep, learning, and memory. However, the key proteins through which the Gas pathway acts to mediate these functions remain poorly understood or simply unidentified. To address this, we have undertaken forward genetic screens to isolate mutants that suppress the strong phenotypes of kin-2(ce179) mutants, containing hyperactivated Protein Kinase A. These mutants exhibit hyperactive locomotion, slow growth, and aldicarb hypersensitivity resulting from overactivation of the Gas pathway in both muscle cells and neurons. In Screen A, we are using the COPAS Biosort to plate 3 adult kin-2(ce179) (F1 progeny of EMS-mutagenized animals) per well on 24-well plates and screening for adult F2 mutants with improved growth and/ or sluggish locomotion. In Screen B, we are plating 200 kin-2(ce179) L1s (F2 progeny of mutagenized animals) per well on 24-well plates containing 0.1 mM Aldicarb and screening for the presence of F3 and F4 progeny after 7d at room temperature. We have nearly completed the screening cycles and have begun crossing the suppressor mutations away from the parent strain, mapping, and complementation analysis. Of special interest are several classes of locomotion-impaired mutants isolated in both screens that do not correspond to previously identified unc genes. Despite the large number of genes known to confer aldicarb resistance, a group of 11 mutants from screen B included only 1 allele of the synaptic vesicle priming protein UNC-13 (which was only borderline resistant to 0.1 mM Aldicarb as a double with kin-2(ce179)), 3 alleles of egl-10 (RGS for Gao), and 7 mutants that do not correspond to known aldicarb resistance loci. The semi-clonal design of screen A allowed us to isolate 10 sterile paralyzed kin-2(ce179) suppressors, 2 of which are null mutants of the Ga GEF RIC-8 and at least one of which is not ric-8.

Carolyn Phillips et al. (2006) Development & Evolution Meeting "A Family of Zinc Finger Proteins is Essential for Meiotic Chromosome Synapsis"

Abby Dernburg, Carolyn Phillips, Jacqueline Hendries

[Development & Evolution Meeting, 2006]

In C. elegans, proper chromosome alignment and synapsis initiation during meiosis are mediated by special regions called "Pairing Centers" that lie near one end of each of the six chromosomes. We have shown that these regions have two separable activities - they stabilize homolog pairing and contribute to initiating synapsis. We previously characterized the protein HIM-8 as an essential trans-acting factor of the X chromosome Pairing Center. It is required for X chromosome pairing and synapsis and localizes specifically to the X chromosome Pairing Center. him-8 mutations had no discernable effect on the segregation behavior of any of the autosomes. him-8 is located in an unusual operon containing three additional genes with high homology to him-8 and each other, which we have named zim-1, -2, and -3 (for zinc finger in meiosis). More recently, we have demonstrated analogous roles for ZIM-1, -2, and -3 at the autosomal Pairing Centers through mutational analysis and immunolocalization. In addition, we have begun to examine the orthologs of ZIM/HIM-8 family in related nematodes. C. remanei, like C. elegans, has four members of this protein family, but C. briggsae, has five, indicating expansion/contraction of this cluster. Recent efforts in our lab have focused on identifying the DNA elements that confer Pairing Center activity. We first showed that HIM-8 protein is recruited efficiently to an extrachromosomal array containing a 30-kb segment of the X chromosome, and have recently whittled this initial HIM-8 recruiting segment down to a 250 bp DNA fragment. Within this short segment are several copies of a 21-bp repeat that is highly enriched over an extensive region on the left end of the X chromosome, relative to the rest of the X chromosome or the autosomes. Based on this candidate sequence, we have examined the genomic regions corresponding to the autosomal Pairing Center regions for similarly enriched sequences. We are currently testing the affinity of these proteins for candidate sequences in vitro, using a fluorescent oligonucleotide binding assay. This group of genes encodes the first chromosome-specific proteins implicated in homolog pairing and synapsis. Analysis of this family of proteins and their chromosomal binding sites is revealing new insights into the mechanisms that bring homologs together to ensure proper segregation.

Patrick C. Phillips (2010) Evolutionary Biology of Caenorhabditis and Other Nematodes "Creating a sexy model: Genetic and genomic resources in C. remanei"

Patrick C. Phillips

[Evolutionary Biology of Caenorhabditis and Other Nematodes, 2010]

Although C. elegans has served as a wonderful model for genetic and functional analysis, it is clear that most of recent and current evolutionary history is dominated by its selfing mode of reproduction, especially from the standpoint of population genetics. A growing number of studies have shown that the closely related gonochoristic, fully outcrossing species C. remanei displays orders of magnitude more molecular and quantitative genetic variation. Indeed, levels of polymorphism within C. remanei exceed those of most animals, providing unique opportunities, as well as serious difficulties (such as profound inbreeding depression). Here, I summarize what is known about the evolutionary genetics of this species and present an update on the efforts that my lab has made in creating a set of canonical recombinant inbred lines, using next generation sequencing to produce a high density genetic map, in fully assembling the genome, and in creating a drug-based transformation system.

Carolyn M Phillips et al. (2005) International Worm Meeting "A family of zinc finger proteins is essential for homolog synapsis"

Chihunt Wong, Carolyn M Phillips, Abby F Dernburg

[International Worm Meeting, 2005]

The first division of meiosis is unlike meiosis II and mitosis, in that each chromosome must recognize and segregate away from its homologous partner, rather than its sister. This division requires a series of coordinated steps that occur during meiotic prophase. Homologous chromosomes must align along their entire lengths, after which proteinaceous structure called the synaptonemal complex polymerizes between the partners. In worms, proper alignment and synapsis initiation depends on cis-acting chromosome regions referred to as Pairing Centers. We have investigated the role of him-8, which encodes a protein specifically required for X chromosome pairing and synapsis. HIM-8 is a C2H2 Zn finger protein that specifically associates with the X chromosome Pairing Center throughout meiotic prophase. In the absence of HIM-8, the X chromosomes fail to synapse, and as a result, they fail to undergo normal recombination and segregation. him-8 is located in an unusual operon containing three additional genes with high homology to him-8 and each other, which we have named zim-1, 2, and 3 (for zinc finger in meiosis). ZIM-1, -2, and -3 each localize to the Pairing Center regions of specific autosomes, and also associate with discrete structures at the nuclear envelope. Detailed characterization of a zim-2 deletion mutant reveals a phenotype very similar to the him-8 mutant phenotype, except that chromosome V, rather than X, is uniquely affected. Chromosome V never achieves a degree of pairing significantly above background premeiotic levels. Consequently, it rarely, if ever, undergoes synapsis, and as a result, there is a high level of meiotic mis-segregation and inviable offspring due to aneuploidy. This group of genes encode the first chromosome-specific proteins implicated in homolog pairing and synapsis. Analysis of this protein family is revealing new insights into the mechanisms that bring homologs together to ensure proper segregation.

Naomi Phillips et al. (2008) Development & Evolution Meeting "Direct Estimate of the Rate and Spectrum of Microsatellite Mutation in Rhabditid Nematodes"

Andy Custer, Matthew Salomon, Gigi Ostrow, Naomi Phillips, Charles Baer

[Development & Evolution Meeting, 2008]

Simple sequence repeats (SSR, or microsatellites) are one of the most widely used classes of genetic markers, from genetic mapping and association studies to investigations of population structure and conservation. Despite their utilization, many unresolved questions concerning the mutational processes shaping SSR evolution remain. Here we describe a study aimed to provide unbiased estimates of the rate and spectrum of new SSR mutations in a novel mutation accumulation (MA) model system. The mutational properties of dinucleotide SSRs were characterized in two strains (= genotypes) of two species in the genus Caenorhabditis, C. briggsae and C.elegans after 250 generations of MA. Mutations have been allowed to accumulate in the (relative) absence of natural selection, thus allowing us to genotype paired control and generation 250 MA lines at approximately 40 dinucleotide loci of perfect, imperfect, and compound SSRs. Furthermore, we investigate how the mutational properties of SSRs are correlated with the genomic mutation rate for fitness in these two species.

Carolyn M Phillips et al. (2003) International Worm Meeting "Identification of cis-Acting Factors Involved in Meiotic Pairing Center Function"

Carolyn M Phillips, Abby F Dernburg

[International Worm Meeting, 2003]

Meiotic cell division is an important process that halves the diploid number of chromosomes to make haploid gametes, so that when fertilization occurs to produce an embryo, diploidy is regenerated. Errors in chromosome segregation are usually lethal in early embryonic development, and in cases where the embryo survives, severe developmental defects are common. The means by which chromosomes segregate properly lies in their ability to successfully contact their homologous partners, align along their entire lengths, synapse, and recombine. Despite much interest in the chromosome dynamics of meiotic prophase, these processes are still not well understood. Previous studies of chromosome rearrangements in C. elegans have shown that particular regions near the end of each chromosome are important for homologous recombination along that chromosome. It has been hypothesized that these regions, currently referred to as Pairing Centers, are not necessary for recognition of homologous partners, but instead stabilize homolog interactions, perhaps by acting as loading sites for factors necessary for recombination or synapsis between homologs. C. elegans is an ideal model system in which to investigate cis-acting components involved in homolog interactions because of the previous genetic studies and because chromosome missegregation defects produce a well-defined phenotype, the High Incidence of Males (Him) phenotype, that will allow ease of scoring for genetic manipulation. There are two approaches that I am taking to uncover the cis-acting elements important for pairing and synapsis. To define the Pairing Center region more precisely, I am doing a forward genetic screen to create more precise deletions of the X chromosome Pairing Center. In parallel, I am attempting to dominantly disrupt the Pairing Center by injection of YACs spanning the known X chromosome Pairing Center region. Both of these experiments will help me define the sequence or properties required for Pairing Center function. Thus far, one novel mutation that specifically perturbs X chromosome Pairing Center function has been identified and is being characterized using cytological and genetic tools.