Vargas, Elizabeth et al. (2017) International Worm Meeting "Trisomy correction occurs during both meiosis I and meiosis II."

Korf, Ian, Friedman, Jacob, McNally, Francis, McNally, Karen, Vargas, Elizabeth, Wang, David

[International Worm Meeting, 2017]

The vast majority of eukaryotes have exactly two copies of each chromosome and reproduce sexually through the process of meiosis. Accurate chromosome segregation requires the physical attachment of exactly two homologous chromosomes by a crossover so that each homologous chromosome can form attachments to opposite poles of a bipolar spindle. Thus a third copy of a chromosome might be expected to segregate randomly, resulting in 50% of progeny inheriting the extra chromosome. Hodgkin et al. (Genetics 91:67) reported that C. elegans with a third copy of the X chromosome have far fewer than 50% trisomic progeny. Cortes et al. (Elife 4:e06056) found that the preferential elimination of the extra X occurs during anaphase I of female meiosis. To test whether extra copies of all C. elegans chromosomes are preferentially eliminated, we scored the inheritance of three different DNA polymorphisms for each autosome among the progeny of triploid hermaphrodites mated with diploid males. For all 5 autosomes, the frequency of trisomic offspring was significantly less than expected from random segregation. Preferential elimination of the extra copy of chromosome V was confirmed by FISH. We also generated a trisomy IV worm strain and when trisomy IV hermaphrodites were mated to diploid males, the frequency of triplo IV progeny was again much lower than expected from random segregation. Cortes et al. (2015) found that univalent X chromosomes do not lose cohesion during anaphase I and thus lag before being preferentially captured by the polar body. Counting of mCherry:histone-labeled chromosomes in triploids revealed that chromosome number decreased between metaphase I and metaphase II and decreased again between metaphase II and diakinesis in the adult progeny. Data from live imaging of anaphase chromosome segregation and REC-8 staining of fixed meiotic spindles support a model in which some univalents remain intact during anaphase I whereas other univalents segregate apart at anaphase I. The resulting single chromatids then lag during anaphase II and are preferentially captured by the second polar body. These data demonstrate that asymmetric female meiotic divisions can correct triploidy and trisomy in C. elegans.

Thomas, Cristel G et al. (2011) International Worm Meeting "Evolution of Sex-biased Expression in the Caenorhabditis genus."

Korf, Ian, Smith, Harold E, Li, Renhua, Thomas, Cristel G, Haag, Eric S, Oliver, Brian

[International Worm Meeting, 2011]

Mating systems shape genome structures, both indirectly through their effects on population genetics and directly due to the genetic control of reproductive traits. Most extant Caenorhabditis species are gonochoristic (male/female), while the most studied species, C. elegans and C. briggsae, are androdioecious (self-fertile hermaphrodite/male). Both selfing species display an overall reduced ability to mate, suggesting that the selective pressure on maintaining efficient mating was weakened as selfing arose. The genes underlying these traits were likely to have been expressed in a sex-biased fashion in the gonochoristic ancestor, and we hypothesized that as selfing emerged their regulation was modified or they were lost altogether. This hypothesis is especially interesting given that selfing species have consistently smaller genome sizes than their gonochoristic relatives. We sought to address whether a disproportionate loss of genes with sex-biased expression accompanies the loss of mating-related traits in Caenorhabditis hermaphrodites. One approach uses RNA-seq to perform a comprehensive survey of adult sex-biased transcripts in C. japonica, C. brenneri, C. remanei and C. elegans fog-2(q71). This allows us to determine the overall number and fraction of sex-biased genes for each species, and hence test the above hypothesis genome-wide. Another approach involves identification and characterization of specific male genes lost in one or more hermaphrodites. Taken together, our results indicate a substantial erosion of reproductive genes has occurred in selfing lineages. We suggest that, with respect to reproductive biology, C. elegans and C. briggsae are actually rather poor models for the rest of Caenorhabditis.

Polinko E et al. (1997) International C. elegans Meeting "The Role of CKS-1 in Embryogenesis"

Strome S, Korf I, Polinko E

[International C. elegans Meeting, 1997]

The cks-1 (cyclin-dependent kinase subunit) gene product has been implicated in regulation of the cell cycle in organisms from yeast to frogs. Although it is known that cks-1 is an essential gene in yeast, whose product physically interacts with both cdk (cyclin-dependent kinase) and cyclins, a clear picture of the exact role of cks-1 in the cell cycle has yet to be uncovered. Our lab obtained a C. elegans cks-1 homolog as a downstream gene in an operon with mes-6 (cloned by Ian Korf). This C. elegans homolog provides an opportunity to analyze the in vivo role of cks-1 in both meiosis and mitosis in a multicellular organism. Toward this end, we have injected antisense cks-1 RNA into the germline of wild-type hermaphrodites and analyzed their progeny. We have analyzed the development of these "antisensed embryos" by Nomarski, as well as by immuno- fluorescence staining of centrosomes, microtubules, and DNA. These embryos show a delay/block in the completion of meiosis and in progression through mitosis. The mitosis defect appears to be an arrest of chromosomes at a stage before anaphase, although the MTOCs continue to duplicate and set up new spindles. Our results support the hypothesis that C. elegans CKS-1 serves an essential role in both meiosis and mitosis, and that the mitosis role may be at the metaphase-to-anaphase transition. We are currently investigating at what point the delays/blocks in meiosis and mitosis occur, and we are attempting to immunolocalize CKS-1 in C. elegans.

Yin, Da et al. (2014) Evolutionary Biology of Caenorhabditis and Other Nematodes "GENOMIC AND FUNCTIONAL CHARACTERIZATION OF GENOME SHRINKAGE IN CAENORHABDITIS"

Meyer, Barbara J., Haag, Eric S., Schartner, Caitlin M., Ralston, Edward J., Korf, Ian, Thomas, Cristel G., Yin, Da, Schwarz, Erich M.

[Evolutionary Biology of Caenorhabditis and Other Nematodes, 2014]

Sexual mode evolves rapidly in some eukaryotic lineages. This is expected to have pronounced consequences for population genetics, sexual differentiation and the nature and intensity of sexual selection, all of which may be reflected in the genome. C. elegans is a self-fertile species, derived recently from an obligately outcrossing male-female ancestor.This trait has evolved in at least two other species of the Elegans sub-genus, C. briggsae and C. sp. 11 (Kiontke et al. 2011). Previous studies indicate that selfing species have smaller genomes and several thousand fewer protein-coding genes than their outcrossing ancestors (Thomas et al. 2012). This reproducibility may be stimulated by an interaction between partial selfing and segregation distortion affecting large indels in male meiosis (Wang et al. 2010). However, the size, location, and gene content of specific deletions remain unknown for any natural system. To characterize the process of genome shrinkage, we have produced a genome assembly from the closest known outcrossing relative of C. briggsae, C. nigoni (formerly C.sp. 9; Felix et al. 2014). The C. nigoni genome is roughly 20 Mb (20%) larger than that of C. briggsae. By comparing C. nigoni contigs with the chromosome-level assembly for C. briggsae, we created an approximation of the C. nigoni physical map. Genome-wide sequence alignment showed the majority of the size reduction is located on the two arms of the five autosomes. Using C. sp.5 as an outgroup, we are able to identify gene family reductions, as well as specific genes recently lost in the C. briggsae lineage. Finally, we present detailed characterization of a family of rapidly evolving proteins that were independently lost in C. elegans and C. briggsae, the MSS (male-specific secreted) family, We have characterized their temporal and spatial expression, and find they are likely to be transferred to the female reproductive tract. We are now developing assays to reveal potential physiological responses to MSS proteins in females, including using calcium imaging to localize putative responder cells in female reproductive tract. References Felix, M.-A., C. Braendle, et al. (2014). "A streamlined system for species diagnosis in Caenorhabditis (Nematoda: Rhabditidae) with name designations for 15 distinct biological species." PLoS One 9: e94723.Kiontke, K., M.-A. Felix, et al. (2011). "A phylogeny and molecular barcodes for Caenorhabditis, with numerous new species from rotting fruits." BMC Evol Biol 11: 339.Thomas, C. G., R. Li, et al. (2012). "Simplification and desexualization of gene expression in self-fertile nematodes." Curr Biol 22: 2167-2172.Wang, J., P. J. Chen, et al. (2010). "Chromosome size differences may affect meiosis and genome size." Science 329: 293.

Papp, Andy et al. (2009) International Worm Meeting "Investigation of Low-cost GFP Microscopy."

Papp, Andy, Aldrich, Chris, Bangalore, Srikanth, Perry, David

[International Worm Meeting, 2009]

The Green Fluorescent Protein (GFP) gene from the fluorescent jellyfish Aequorea victoria, and its variants (such as EGFP), are used extensively to study the location and timing of gene expression in transgenic animals and plants (Chalfie et al, 1994). Resultant GFP fusion proteins are especially well-suited to studying in vivo gene expression patterns in the transparent nematode, C. elegans and the transparent larva of the fly D. melanogaster. Perhaps one of the most significant limitations to its use in large-scale genetic screens is the high cost of equipment needed to observe GFP microscopically - namely the Fluorescence Dissecting Stereomicroscope for screening and picking mutants and upright and inverted epi-fluorescent compound microscopes for more detailed studies. Commercially available Fluorescence Dissecting Stereomicroscopes typically sell in the midrange between US$10,000 and US$20,000. They are offered by only the "high-end" microscope companies, based upon their most expensive dissecting scopes, and incorporate their premium-priced mercury arc-lamp illuminators, power supplies and epi-fluorescence modules. This leads us to wonder whether it is possible to cut corners without sacrificing utility, and which corners can be cut. Since the inception of GFP-based expression screens, a variety of researchers have produced custom-made and home-made GFP dissecting scopes. These include, for example, Welcome Bender (Harvard Medical School, pers. comm.) and Ian Chin-Sang (Chin-Sang, 2004). There are several possibilities to consider toward lowering the cost of a Fluorescence Dissecting Stereomicroscope. It may be possible to get sufficient light from relatively inexpensive, long-lived, lower-power-consuming Light Emitting Diodes (LEDs) vs. mercury arc lamps. Depending upon the spectral specificity of the LEDs, filters or dichroic mirrors might be omitted. Getting enough light intensity of the precise wavelengths that excite GFP fluorescence focused onto the sample is important. The exciting beam can be provided directly or focused backward through the microscope using "epi-illumination". We will explore these possibilities and report (and possibly demonstrate) the results. Chin-Sang, Ian. (2004) GFP Stereoscope Using LED light source. Queen''s University, Kingston, ON, Canada. http://130.15.90.245/gfp_stereoscope.htm.

Xu L et al. (1997) International C. elegans Meeting "OPERONS WITH NO DNA BETWEEN THE GENES"

MacMorris MA, Xu L, Blumenthal TE

[International C. elegans Meeting, 1997]

Analysis of the C. elegans genome indicates that approximately 25% of genes are in operons with 100-300 bp between them. However, we are now aware of three instances of gene pairs without any DNA between them. These include one published example,cyt-1:ced-9 (Hengartner and Horvitz, 1994), one discovered by Susan Strome and Ian Korf (submitted) mes-6:cks-1, and a new example we found on a sequenced cosmid, which includes the U170K gene and an unidentified upstream ORF. In each of these gene pairs, the polyadenylation signal, AAUAAA, of the upstream gene is within a few bp of the trans-splice site for the downstream gene, and the 3' ends of the upstream genes map to the nucleotide adjacent to the trans-splice sites of the downstream genes. Furthermore, whereas in the usual operons downstream genes are primarily trans-spliced to SL2, in these 3 examples the downstream genes are exclusively SL1 trans-spliced. We believe these three represent a new class of operon in which SL1 trans-splicing is the sole event that creates the monocistronic mRNAs, and in which the SL1-dependent cleavage leaves a free 3' end on the upstream mRNA which is then polyadenylated by the normal mechanism. To test this idea, we tried creating such an operon by moving the AAUAAA of the standard type operon, gpd-2:gpd-3, to a location adjacent to the trans-splice site of the downstream gene,gpd-3. In vivo processing of the RNA from this construct was tested in transgenic worms. Unfortunately, this simple change was not sufficient to change the operon to the novel type. Although all 3' end formation now occurred just downstream of the new AAUAAA location, it was not 100% dependent on trans-splicing. Furthermore, a mixture of SL1 and SL2 trans-splicing was observed in this experiment. In addition we have studied the mes-6:cks-1 operon in more detail. Remarkably, a third putative gene within the 3' UTR of mes-6 is created by SL1 trans-splicing at both ends: at its 5' end at a site within the penultimate exon of mes-6, and at its 3' end at the 5' end of cks-1. Thus SL1 trans-splicing appears to be responsible for the key RNA processing events that occur at both ends of this RNA.

Biron RW et al. (1996) East Coast Worm Meeting "gon-2: Expression and Function by Inference."

Sun Y-a, Biron RW, Lambie EJ

[East Coast Worm Meeting, 1996]

The somatic gonad of C. elegans develops entirely from the postembryonic blast cells, Z1 and Z4. gon-2 is required for the division and differentiation of these cells. All of the alleles of gon-2 that we have isolated cause a partial maternal-effect gonadless (Gon) phenotype. Tissues other than the somatic gonad appear unaffected. The degree of gonad development is variably penetrant for all alleles; in some animals, Z1 and Z4 fail to divide at all. Cell lineage and pes-1::lacZ expression studies in Gon animals suggest that gon-2 acts to regulate the timing of division of Z1, Z4 and their descendants (thanks to Ian Hope for pes-1::lacZ). q388 is temperature sensitive, and has a more penetrant phenotype than any of the other alleles. The TSP of the Gon phenotype of q388 suggests that gon-2 translation may begin early in embryogenesis, well before the birth of Z1 and Z4. We are currently using a cosmid that rescues gon-2 for mosaic analysis. At the moment, the genome project ftp site is only one cosmid away from gon-2. So, by the time of the meeting we should have some idea about the possible sequence of the gene.

Jane Shingles et al. (2006) European Worm Meeting "Progress of the Localization of Expression Mapping Project"

Jane Shingles, Denis Dupuy, Marc Vidal, Ian Hope, John Reece-Hoyes

[European Worm Meeting, 2006]

Jane Shingles1, John Reece-Hoyes1, Denis Dupuy2, Marc Vidal2 and Ian Hope1. The Promoterome library containing 6500 Caenorhabditis elegans promoter fragments has previously been generated and used to construct promoter ::GFP fusions to allow determination of gene expression patterns in C. elegans. Optimization of the C. elegans micro-particle bombardment transformation technique, to give a medium through-put system, allowed the generation of expression patterns for over 300 C. elegans promoter::GFP fusions. Promoter fusion constructs of transcription factors genes and genes with homology to human genes with no assigned function were selected for the initial analysis and the results are shown. Initial analysis of the results highlights several significant differences between the two sets of genes. Promoters from C. elegans homologues of human genes with no known function were far more likely to yield either no GFP expression (35% vs. 6%) or ubiquitous GFP expression (23% vs. 8%) under the standard laboratory conditions utilized. In contrast, promoters from C. elegans transcription factor genes were far more likely to drive neuronal expression (62% vs.16%).. Full results are presented on the Hope Laboratory Expression Pattern database URL:http://bgypc059.leeds.ac.uk /~web

Francesca Palladino et al. (1997) International C. elegans Meeting "Towards the isolation of a cec-1 mutant"

Fritz Mueller, Francesca Palladino

[International C. elegans Meeting, 1997]

The chromo domain was originally identified in Drosophila as a sequence motif common to the Polycomb repressor protein (PC) and the heterochromatin-associated protein HP1. Other proteins containing this motif have subsequently been identified in a number of species from plants to mammals. This conserved domain appears to be common to chromatin-associated proteins. The C. elegans gene cec-1 encodes a chromo domain-containing nuclear protein present in all somatic cells from the 50-80 cell-stage onwards to adult animals. CEC-1 protein is not detected in early embryos and germ cells, while cec-1 mRNA is present in all blastomeres of the early embryo. Immunolocalization experiments show a homogeneous distribution of CEC-1 within interphase nuclei. The expression pattern of CEC-1 suggests that it represents a novel chromosome-associated protein in C. elegans. (Agostoni et al. 1996) In order to study the function of cec-1, we are attempting to isolate a mutation in this gene. A Tc1 insertion was isolated in the laboratory of Dr. Ian Hope (Leeds University) from which we are trying to isolate a deletion derivative. As a parallel approach, we are also testing the possibility that the cec-1 coding region rescues candidate lethal mutations in transgenic strains.

Karishma Sadikot et al. (2006) Development & Evolution Meeting "A Novel Dosage Compensation Protein with Chromosome Segregation Functions"

Karishma Sadikot, Barbara Meyer, Nizar Taki, Gyorgyi Csankovszki, Emily Petty

[Development & Evolution Meeting, 2006]

The C. elegans dosage compensation complex (DCC) assembles on hermaphrodite X chromosomes to downregulate gene expression two-fold. This ensures equal amounts of X-linked gene products in XX hermaphrodites and XO males. The DCC is similar to the 13S condensin complex, which is essential for mitotic and meiotic chromosome segregation. The four known DCC subunits are homologous to four of the five condensin proteins. In search of the fifth DCC subunit, antibodies against the unique dosage compensation protein, DPY-27, were used to immunoprecipitate the DCC from embryonic extracts. In collaboration with Ian McLeod and John R. Yates III, MUD-PIT analyses of precipitated proteins identified a novel DCC subunit, the protein encoded by F29D11.2. PSI -BLAST identified F29D11.2 as the homolog of the condensin subunit CAP-G. Using RNAi, we confirmed that this protein plays a role in dosage compensation. F29D11.2 is required for localization of other DCC subunits, and it will only localizes to the X in the presence of all other subunits, indicating that the protein is an integral member of the worm DCC. Interestingly, F29D11.2 appears to also interact with the worm mitotic condensin. Our preliminary results indicate that the protein is present in immunoprecipitated mitotic condensin complexes. The protein also appears to localize to germline nuclei. Furthermore, animals homozygous for a null mutation in F29D11.2 arrest during larval development with chromosome segregation defects, including chromatin bridges connecting interphase nuclei. These results suggest that F29D11.2 may function in both dosage compensation and mitotic and meiotic chromosome segregation.