Chung, George et al. (2011) International Worm Meeting "A member of the DOG-1 family is essential for normal cell division."

Rose, Ann, Chung, George

[International Worm Meeting, 2011]

The Rose laboratory has been studying genes encoding members of a helicase subfamily that includes dog-1 (1,2 and see poster by Jones & Rose, this meeting) and rtel-1 (3,4). A third member of this family has orthology to the yeast gene, CHL1. In the absence of the gene function, worms grow to become sterile adults (Ste) with a movement defect (Unc). Using the D-neuron-specific GFP marker (unc-47::gfp, constructed by the Jorgensen lab), we visualized these neurons with fluorescence microscopy. Both newly hatched wild-type and mutant animals showed 6 DD neurons derived from embryogenesis. Additional 14 D neurons (13 VD and DVB) developed in the wild-type animals prior to adulthood. In mutants, however, we observed an average of only 12 D neurons rather than the expected 20. In order to determine if other cell types were affected in a similar manner, we used a seam cell marker (scm::gfp, constructed by the Rothman lab) to visualize the presence of seam cells during L4. The homozygous mutant worms only had an average of 10 seams cells per row, fewer than the 16 in wild-type worms. Furthermore, many mutant animals did not have mature oocytes. In animals that did have mature oocytes, DAPI staining revealed diakinetic chromosome spots in excess of the expected six. Taken together, this gene is requireed for cell division. (1)Youds, J.L., O'Neil, N.J. & Rose, A.M. Homologous recombination is required for genome stability in the absence of DOG-1 in Caenorhabditis elegans. Genetics 173, 697-708 (2006). (2)Youds, J.L. et al. DOG-1 is the Caenorhabditis elegans BRIP1/FANCJ homologue and functions in interstrand cross-link repair. Mol. Cell. Biol 28, 1470-1479 (2008). (3)Barber, L.J. et al. RTEL1 maintains genomic stability by suppressing homologous recombination. Cell 135, 261-271 (2008). (4)Youds, J.L. et al. RTEL-1 enforces meiotic crossover interference and homeostasis. Science 327, 1254-1258 (2010).

Chung, George et al. (2013) International Worm Meeting "CHL-1 is required for DNA replicative integrity in Caenorhabditis elegans."

Rose, Ann M, Chung, George

[International Worm Meeting, 2013]

Previously, we have described chl-1 in C. elegans, an ortholog of the human DDX11/ChlR1 gene associated with Warsaw breakage syndrome. Our published results have shown that CHL-1 is indispensable for cell proliferation and for genome integrity. Using the continuously-replicating, mitotic nuclei of the C. elegans germline, we have begun to assay the recruitment of repair factors to the DNA under various non-damaging and damaging conditions. Even in the absence of DNA damaging agents, the chl-1 mutant DNA is marked by an abnormally high number of RAD-51 foci, indicating that the CHL-1 protein is required for the normal, breakage-free completion of mitotic replication.

Jones, Martin R. et al. (2011) International Worm Meeting "Genetic and molecular investigation of the Fanconi Anemia pathway in C. elegans."

Jones, Martin R., Rose, Ann M.

[International Worm Meeting, 2011]

Fanconi Anemia (FA) is a rare autosomal recessive cancer susceptibility syndrome associated with various congenital abnormalities and a predisposition to developing various types of cancers. The Fanconi pathway prevents chromosome instability by resolving DNA lesions that block replication and transcription, such as interstrand crosslinks (ICL). It is thought that the FA pathway acts to coordinate the repair of DNA damage through different DNA repair effectors such as the Homologous Recombination repair (HR), Nucleotide Excision Repair (NER), and Translesion Synthesis (TLS) pathways. C. elegans has a simplified Fanconi Anemia pathway which has proven to be a valuable model for the discovery and investigation of conserved FA-associated genes. Our lab previously identified DOG-1 as the functional orthologue of the human FANCJ helicase. Animals mutant for a null allele, dog-1(gk10), share many of the hallmarks of FA cells such as sensitivity to interstrand crosslinks (ICLs). Using a combination of genetic and molecular techniques we are exploiting C. elegans genetics to investigate the role of FA-associated genes in maintaining genome stability. We are combining this approach with a variety of screening strategies to identify additional gene functions that when disrupted exacerbate the genomic instability of animals defective for FA-associated genes. We have identified a number of genetic interactions with dog-1(gk10) mutants using the increased frequency of small G-tract deletions that occur in the absence of DOG-1 function as an assay. These include homologous recombination repair components, translesion synthesis (TLS) polymerases, and the Fanconi anemia pathway gene fcd-2/FANCD2. In addition, we have examined the viability of dog-1(gk10) animals with other mutants, revealing interactions with the RecQ helicase HIM-6/BLM, which is implicated in the resolution of stalled replication forks, and the anti-recombinase RTEL-1/RTEL, which results in synthetic lethality. The variety of genetic and molecular tools combined with a conserved, though simplified, FA pathway makes C. elegans an excellent animal model in which to identify new FA-associated genes and to elucidate the function of the FA pathway.

Tam, Annie et al. (2013) International Worm Meeting "Molecular characterization of mitocycin C-induced lethal mutations in Caenorhabditis elegans."

Chu, Jeffrey SC., Rose, Ann M., Tam, Annie

[International Worm Meeting, 2013]

Mitomycin C (MMC) is a DNA crosslinking agent used clinically as a diagnostic for the genome instability syndrome Fanconi anemia, and also as a chemotherapeutic agent to treat a wide range of cancers. MMC covalently interacts with guanines in GC-rich segments of DNA, resulting in inter- and intrastrand DNA crosslinks, as well as DNA adducts. These interactions can cause DNA lesions that inhibit critical cellular processes such as replication, and if unrepaired lead to a range of chromosomal lesions. Although widely used, the prevalence and range of genomic instability caused by MMC has not been characterized. The nematode Caenorhabditis elegans is a well characterized genetic model in which to study genomic damage generated by exposure to crosslinking agents. In this context, we have performed forward genetic screens, determined the forward mutation frequency at different doses of MMC, and have isolated a number of lethal mutations for study.The lethal mutations were isolated and maintained as heterozygotes using hT2, a genetic balancer involving a reciprocal translocation between chromosomes I and III. We used three-factor mapping to narrow down the location of over 60 MMC-induced lethal mutations on chromosomes I and III. DNA from the genetic strains carrying the MMC-induced mutations has been sequenced using next-generation sequencing. As a consequence of the precise mapping of the lethal mutations, we can efficiently identify the physical basis of the lesions. We are in the process of characterizing the spectrum of mutagenic lesions caused by MMC, examining both their nature and frequency. Identification of the types of DNA lesions caused by MMC not only furthers our understanding of this widely used drug, but may also lend insight into the molecular mechanisms required for their repair.

Stewart HI et al. (1996) West Coast Worm Meeting "Rescue of New Essential Genes on LGIII(left), in the Nematode Caenorhabditis elegans."

Stewart HI, Franz NW, Barbazuk BW, Baillie DL, Ha TT

[West Coast Worm Meeting, 1996]

We utilized trangenic strains containing cosmids, sequenced by the sequencing consortium, to rescue the lethal phenotype of several essential genes (See presentation by Diana Janke et al, this session). The rescued genes are positioned on LGIII(left) balanced by the duplication sDp3(see poster; Stewart et al, this session). A set of overlapping deficiencies was utilized to define areas within which to focus our rescue attempts). PCR analysis was utilized to further define the end points of these deficiencies, facilitating our analysis ( see poster ; Norm Franz et al, this session). We concentrated our analysis on areas to the right of dpy-17, in the interval between dpy-17 to dpy-19. Lethals mapping to sDf125 that were not rescued by sDp8 are presented in a poster by Nigel O'Niel et al, this session. We have rescued several essential genes. Our rescues will facilitate the functional analysis of these new genes. These rescues have also helped us to align and correlate the physical and genetic maps. Our estimates are that there will be more than two essential genes per cosmid. A total of 202 recessive lethal mutations have been generated, using EMS as a mutagen. We intend to make molecular assignments of all of these elements, through our cosmid rescue experiments. As we have estimated that there is only a 28% saturation of the area for essential genes, we will be generating more recessive lethal genes, to facilitate this analysis. This work has been funded by grants from the National Institute of Health (N.I.H.), U. S.A. to Ann Rose; D. Riddle and David Baillie and Canadian Genome and Advanced Technologies (C.G.A.T.), Canada to Ann Rose and David Baillie.

Jones, Martin R. et al. (2013) International Worm Meeting "Characterisation of genomic instability and interstrand crosslink sensitivity associated with mutation of dog-1, the functional ortholog of human Fanconi Anemia protein FancJ."

Rose, Ann M., Chu, Jeffrey S., Jones, Martin R.

[International Worm Meeting, 2013]

C. elegans has a simplified FA pathway and as such has proven to be an invaluable model for the discovery and investigation of conserved FA-associated genes. We have previously identified DOG-1 as the functional ortholog of the human FANCJ helicase. Animals mutant for a null allele, dog-1(gk10), share many of the hallmarks of FA cells such as increased genomic instability and sensitivity to interstrand crosslinks (ICLs). Although our understanding of DOG-1/FANCJ is growing, its precise function in ICL repair remains elusive. We are exploiting C. elegans genetics in combination with whole-genome sequencing (WGS) to investigate the relationship between genome stability and ICL sensitivity in DOG-1/FANCJ deficient animals. We have sequenced genomes derived from a sequential line of animals that have accrued mutations over a defined number of generations under standard laboratory conditions. From this analysis we are able to identify copy number variations (CNVs), including small deletions, and single nucleotide variations (SNVs). In the dog-1(gk10) genomes we find an accumulation of deletions in g-rich DNA as expected. We are currently expanding on this analysis to assess the genomes of dog-1 mutants that have been treated with a variety of mutagenic agents including those known to induce ICLs. We have demonstrated the feasibility of using WGS to assess genomic instability in dog-1 genomes to a high resolution. Assessing the extent and type of genomic damage in ICL treated genomes will lead to a better understanding of how dog-1/FANCJ's function in maintaining g-rich DNA is correlated with its ability to prevent sensitivity to ICL inducing agents. Our findings have potential significance for the informed use of chemotherapeutic agents against cancers arising in FANCJ patients.

Jones, Martin R. et al. (2013) International Worm Meeting "ROL-3, the ortholog of the human proto-oncogene ROS1, is required to orchestrate the morphogenesis and development of the seam syncytium and interacts with the Bicaudal-C homolog bcc-1."

Rose, Ann M., Jones, Martin R., Baillie, David L.

[International Worm Meeting, 2013]

The orphan receptor ROS1 is a human proto-oncogene, mutations of which are found in an increasing number of cancers. Little is known about the role of ROS1, however in vertebrates it has been implicated in promoting differentiation programs in specialized epithelial tissues. Using a combination of genetic and molecular techniques we show that the C. elegans ortholog of ROS1, the receptor tyrosine kinase ROL-3, has an essential role in orchestrating the morphogenesis and development of the seam syncytium, a specialized epidermal tissue, highlighting a potentially conserved function for ROL-3 in coordinating crosstalk between developing epithelial cells. We have also used a deletion biased mutagenesis screen to identify two suppressors of rol-3, providing evidence of a direct relationship between ROL-3, the mucin SRAP-1, and BCC-1, the homolog of mRNA regulating protein Bicaudal-C. Our work answers a long standing question as to the developmental function of ROL-3, identifies three new genes that are expressed and function in the developing epithelium of C. elegans, and introduces the C. elegans as a potentially powerful model system for investigating the increasingly important, yet poorly understood, human oncogene ROS1.

O'Neil NJ et al. (1996) West Coast Worm Meeting "SYSTEMATIC HIGH RESOLUTION MAPPING OF ESSENTIAL GENES IN THE sDf125 REGION BY TRANSGENIC RESCUE."

Kuervers LM, Baillie DL, Vatcher GP, O'Neil NJ

[West Coast Worm Meeting, 1996]

We have demonstrated that transgenic rescue is an effective method for high resolution mapping of essential genes. Our lab is constructing a map of essential genes on LGIII left. We have divided the left end of the central cluster into manageable regions which are defined by overlapping deficiencies and duplications. These regions are determined physically by PCR testing the deficiencies to determine endpoints (see N.Franz et al. abstract, this meeting). Into these regions, we have mapped a set of EMS induced lethals and maternal effect lethals. (see abstracts by H. Stewart et al. and G. Vatcher and D. Baillie, this meeting). One such region is defined by sDf125 and sDp8. This region spans 340 000 base pairs and is completely covered by nine cosmids. Within this region there are 75 putative genes predicted by Genefinder. We have used a sytematic approach to rescue essential genes within the sDf125 region with transgenic cosmids from the library created by J. Schein, D. Janke and The Ha as part of a CGAT grant to Ann Rose and David Baillie (see abstracts by J.Schein et al. and D. Janke et al., this meeting) To date this method has proven very successful. At least 8 genes have been completely rescued in this region by this approach. These transgenic rescues map these genes to 40 kB (one cosmid) resolution as opposed to the 340 kB resolution of the deficiency and duplication data. This is almost a ten-fold increase in resolution and reduces the number of ORFs which may be responsible for the gene function. The next step will be the subcloning of cosmids with which we have rescued essential genes. This will directly corellate the lethal or maternal effect lethal phenotype to the appropriate ORF. We have already begun the subcloning of cosmid C05D11 (see G.Vatcher and D.Baillie abstract, this meeting). This project is funded by a CGAT grant to Ann Rose and David Baillie.

O'Neil NJ et al. (1997) International C. elegans Meeting "ANALYSIS OF THE sDf127 REGION BY TRANSGENIC RESCUE."

O'Neil NJ, Vatcher GP, Kuervers LM, Baillie DL

[International C. elegans Meeting, 1997]

We have demonstrated that transgenic rescue is an effective method for high resolution mapping of essential genes. Our lab is constructing a map of essential genes on LGIII left. We have divided the region between dpy-17 and unc-36 into manageable regions that are defined by overlapping deficiencies and duplications. These regions are determined physically by PCR testing the deficiencies to determine endpoints. Into these regions, we have mapped a set of EMS induced lethal and maternal effect lethal mutations (See abstract H.Stewart et al., this meeting). One such region is defined by sDf127 and sDp8. This region spans 340 000 base pairs and is completely covered by ten cosmids. Within this region there are 75 putative genes predicted by Genefinder. To date, we have mapped 14 essential genes into this region. We have used a sytematic approach to rescue these essential genes with cosmid transgenics from the library created as part of a CGAT grant to Ann Rose and David Baillie (See abstract by D. Janke et al., this meeting) To date this method has proven very successful. At least 8 genes have been completely rescued in this region utilizing this approach. These transgenic rescues map these genes to 40 kB (one cosmid) as opposed to the 340 kB resolution of the deficiency and duplication data. This is almost a ten-fold increase in resolution and reduces the number of coding sequences which may be responsible for the gene function. The next step will be to subclone the cosmids with which we have rescued essential gene mutations. This will directly corellate the lethal or maternal effect lethal phenotype to the appropriate coding sequence. We have already begun the subcloning of the cosmids C05D11 (see abstract by G.Vatcher et al., this meeting), C31H11 and C16A3. This project is funded by a grant from the Medical Research Council of Canada to Ann Rose and David Baillie.

Helen Caspersson et al. (2003) International Worm Meeting "The forkhead transcription factor F26B1.7 plays a role in the mesoderm differentiation."

David Baillie, Ann Rose, Helen Caspersson, Marika Hellqvist-Greberg

[International Worm Meeting, 2003]

The forkhead transcription factors have a conserved DNA-binding domain and they take part in developmental processes in many vertebrates. In C. elegans the forkhead proteins play an important role in embryogenesis, development and aging. The forkhead gene F26B1.7 is a member of the FoxF subfamily of forkhead domain proteins and has a probable role in mesoderm differentiation. The genetic mutation to F26B1.7 is called let-381 and was isolated by the Ann Rose Lab. let-381 is a lethal mutation that causes the worms to die as embryos or as early L1-larvae and a few percent of the worms manage to grow up to adults that often is sterile. When F26B1.7 is silenced by feeding RNAi defects in the mesodermal cell lineage can be seen in adult worms. These worms have reduced number of coelomocytes and the positioning of the SM descendants are affected. By using methods like RT-PCR, GFP reporters and RNAi we are analyzing potential target genes for F26B1.7 to better understand its function during development and differentiation.