Singh, Akanksha (2021) International Worm Meeting "CRISPR- Nanobodies from C. elegans as an therapeutic approach for Erythroblastosis Fetalis"

Singh, Akanksha

[International Worm Meeting, 2021]

Erythroblastosis fetalis is a consequence of incompatibility amongst Rh-negative antigen from mother and Rh-positive antigen of foetus thus resulting in hemolysis. The hemolysis usually results in respiratory problems, neurological disorders or even heart failure. C.elegans is known to be homologous to some extent with human genome and produces monoclonal antibodies. The nanobodies from C. elegans are engineered with CRISPR Cas 9 technology where Cas 9 protein has been incorporated into nanobodies that will accelerate its efficiency. The engineered nanobodies will target genes like RHD and RhCE that are prominent in RBC membranous environment. The nanobodies with Cas-9 might block the interaction of Rh-negative antibodies with Rh-positive antibodies of foetus as by refusing the recruitment of Rh-negative antibodies in placenta. Antibodies from the C. elegans are extracted and engineered to produce nanobodies with encoded Cas 9 protein. The haemoglobin obtained from foetus are mixed with haemoglobin from mother with Rh-antigens in vitro. After a period of 24-48 hours, when engineered nanobodies are treated with blood by ELISA technique indicated reduced count of Rh-positive antibodies thus blocking RHD gene not allowing the interaction with Rh-negative antibodies. The aim of the study was to study molecular interaction occurring after exposure of nanobodies to Rh- antigens present in foetal blood. Keywords: Erythroblastosis fetalis, Rh-negative, hemolysis, C. elegans, CRISPR Cas 9, RHD , RHCE, haemoglobin, nanobodies.

Flavia I Pellerone et al. (2003) International Worm Meeting "Investigation of the functional role of Rh proteins in the nematode Caenorhabditis elegans."

Susan M Howitt, Flavia I Pellerone, Carolyn A Behm, Inge J Stewart

[International Worm Meeting, 2003]

Rhesus (Rh) factors have been extensively studied as blood group antigens. Recently it has become evident that Rh proteins share ~20% homology with methylamine permeases (mep) and ammonium transporters (amt) of yeast, bacteria and plants. Non-erythroid Rh proteins, expressed in kidney and liver, have also recently been identified in mammals. This implies that Rh proteins may function as the elusive ammonium transporter in animals. Putative Rh homologues also occur in lower organisms (green algae, slime mold) and invertebrates such as Caenorhabditis elegans and Drosophila melanogaster. Thus Rh proteins have functions not restricted to red blood cells. The model nematode C. elegans expresses four amt genes, plus two Rh genes (rhr-1 and rhr-2) that share significant identity with human RhAG genes. The considerable biological resources of C. elegans provide an excellent tool for studying the functions of these genes. To test whether the Rh proteins transport ammonium we have commenced complementation studies in a yeast strain lacking all three native ammonium transporters. In initial experiments, rhr-2 cDNA failed to complement but further work is under way. We will also undertake functional characterization of the Rh proteins by expressing them in Xenopus oocytes. We have used RNA interference to investigate the function of these proteins. Silencing expression of rhr-1 and rhr-2, individually or together, reduced mRNA expression by ~80% but induced no obvious phenotype in C. elegans. However, two dimensional gel electrophoresis of protein extracts from these worms has revealed differentially expressed proteins, which are undergoing further analysis.

Polinko E et al. (1995) International C. elegans Meeting "WHAT'S NEW WITH SPE-11, A PATERNAL-EFFECT LETHAL GENE IN C. ELEGANS"

Turner RH, Polinko E, Browning H, Strome S

[International C. elegans Meeting, 1995]

To gain insights into paternal contributions to the embryo, we are analyzing the paternal-effect embryonic-lethal gene spe-11 in C. elegans. Fertilization of wild-type oocytes by sperm from spe-11 mutant worms leads to inviable embryos. In contrast, spe-11 mutant oocytes produce normal embryos when fertilized by wild-type sperm. The spe-11 gene encodes a novel protein that is rich in acidic and basic residues and becomes localized to a perinuclear ring in mature sperm. SPE-11 probably functions in the embryo, since animals engineered to supply SPE-11 to the embryo through the oocyte rather than through the sperm produce viable offspring. To identify regions of SPE-11 that are important for its function, we have: 1) analyzed the four mutant alleles of spe-11, and 2) isolated and sequenced spe-11 homologues from C. briggsae and C. vulgaris to identify conserved regions. The allele sequences, along with protein staining and E.M. of mutant sperm, suggest that SPE-11 is a component of and helps organize the electron-dense halo seen around the nuclei of mature sperm. A similar electron-dense halo and SPE-11 staining pattern are observed in mature sperm from C. vulgaris and C. briggsae.

Tanaka, R. et al. (2013) International Worm Meeting "Quiescence of entomo-phoretic nematode Caenorhabditis japonica."

Kanzaki, N., Hirooka, Y., Kikuchi, T., Tanaka, R.

[International Worm Meeting, 2013]

Entomo-phoretic nematodes are not parasitic to insects but use insects for their transportation. Caenorhabditis japonica, a bacteria-feeding nematode, has a species-specific phoresy with a shield bug, Parastrachia japonensis, and its lifecycle is synchronized to the bug's life. The active dauer larvae (DL) show intensive host-seeking, high sensitivity to oxidative stress and less than 15 days of longevity without attaching the bug. On the other hand, quiescent DL on the bug survive more than 11 months. Thus, the quiescence associated with the bug seems an essential factor for nematode survivability. In the present study, survivabilities of quiescent and active DL were compared under several different conditions to examine the involvement of the bugs in nematode's longevity. Then transcripts and proteins were analyzed to compare gene expression between quiescent and active DL. The quiescent DL on the bug and active DL were kept in a container with 85% (dehydrated condition) or 97% (lightly dehydrated condition which immobilize the DL) of relative humidity (RH). The most active DL died in one week because of desiccation (85% RH) or fungal infection (97% RH). On the other hands, quiescent DL on the bug showed significantly higher survival rate under the same conditions. Further, the survivability of surface-sterilized active DL kept in 97% RH was almost the same as quiescent DL. Therefore, the bugs are likely to work as the shelter from dehydration, and are also providing anti-microbe activity to DL. The expressed genes and proteins also differed between quiescent and active DL. Expression of genes and proteins involved in several stress resistance, metabolic regulation and cuticle formation were significantly higher in quiescent DL. On the other hand, expression of genes and proteins involved in several metabolic related (activity regulation) were significantly higher in active DL. Our results suggest C. japonica DL use their host bug not only for transportation, but also as shelter from environmental stresses and microbe infection. Further, the quiescent stage-specific gene expression allows the extraordinary longevity of this stage.

Nettell JJ et al. (1998) European Worm Meeting "EXPRESSION PATTERN OF C. ELEGANS RH (RHESUS) HOMOLOGUES -AN ATTEMPT TO ASSIGN FUNCTION TO HUMAN RH PROTEINS"

Nettell JJ, Tanner MJ, Kuwabara PE

[European Worm Meeting, 1998]

Rh antigens are highly immunogenic in man and are important in clinical haematology and blood transfusions. They form the major cause of haemolytic disease of the newborn in caucasian populations. The antigens are expressed on two 30kDa non-glycosylated membrane proteins (Rh30) and complex with a glycoprotein (Rhgp) of very close homology. The function of these erythroid specific proteins is unknown. In an attempt to elucidate the function of Rh we have been studying two recently discovered Rhgp homologues in the nematode C. elegans (CERH1/2). We have begun to localise the expression of both genes by constructing GFP-tagged promoter vectors containing a nuclear localisation signal (NLS). The gonad of N2 hermaphrodite C. elegans was injected and double rol-6/GFP transformed worms were visualised by laser confocal microscopy. GFP fluorescence, resulting from CERH promoter activity, indicated that expression predominates in seam cells and in some of the large polyploid nuclei of the intestine. The results also suggest CERH1 and CERH2 co-localise in these cell types. Further work should confirm this co-localisation and determine protein targeting by protein-GFP fusion constructs. Immunocytochemistry, using prepared anti-CERH peptide antibodies, will further investigate such expression patterns.

Siddiqui SS (2000) East Coast Worm Meeting "Kinesin motors moving chromosomal cargo"

Siddiqui SS

[East Coast Worm Meeting, 2000]

Kinesins form a large family of microtubule based motors, which mediate intracellular transport including vesicle delivery, ciliary growth, and chromosomal movement during cell cycle. We have brought to light genes encoding for all known kinesin subfamilies found in mammals in the nematode C. elegans (viz. klp-1-klp-20), by a combination of genetic and molecular approaches. The simple nematode provides an excellent model to elucidate the relationship between structure and function for all kinesin motors within a simple model animal. Our results suggest that the nematode kinesins perform highly specialized cellular functions in ciliary and axonal growth, neurotransmitter vesicle transport, and chromosomal movement during meiosis and mitosis. Most of these in vivo functions are conserved in species as divergent as the nematode and humans. Elucidating kinesin motor function during cell division will unravel how the spindle precisely segregates chromosomes, and may offer insights into the molecular basis of disease states that arise from abnormal spindle dynamics. For example, chromosome non-disjunction during meiosis causes defects as Klinefelter, Down, and Turner Syndromes. Chromosome non-disjunction during mitosis is critical for tumor progression. Chromosome specific kinesin motors provide novel targets for intervention into cell division cycle, and strategies that allow specific blockage of motor function during mitosis may have powerful chemotherapeutic potential.

Aisa Nakashima et al. (2008) European Worm Meeting "Two new germ cell immortality mutants"

Jan Laroque, Theresa M Zucchero, Julie Hall, Aisa Nakashima, Shawn Ahmed, Lauren Garwood

[European Worm Meeting, 2008]

Germ cells represent canonical stem cells that possess the remarkable. quality of being able to proliferate from one generation to the next,. indefinitely, free of replicative damage. mortal germline mutants initially. display normal levels of fertility, but become progressively sterile when. grown for multiple generations. Of 16 original mortal germline mutants (1),. most were temperature-sensitive and only became sterile when grown at 25oC.. Of the ts mutants, two were resistant to RNAi feeding constructs targeting. embryonic lethal genes at all temperatures: 1c and 10i. The 10i mutation. mapped to the right arm of chromosome IV, between unc-22 and dpy-4, and. fails to complement rsd-2 for its RNAi resistance phenotype. rsd-2 has been. reported to be is deficient for spreading of dsRNA-mediated RNAi from. somatic cells to germ cells (2), suggesting that this function may be. relevant to maintaining germ cells over multiple generations during growth. at high temperatures. The mechanism by which this occurs is being studied.. (1) Ahmed S, Hodgkin J (2000). MRT-2 checkpoint protein is required for. germline immortality and telomere replication in C. elegans. Nature.. (2) Tijsterman M, May RC, Simmer F, Okihara KL, Plasterk RH (2004). Genes. required for systemic RNA interference in Caenorhabditis elegans. Current. Biology.

K Brouwer et al. (2004) European Worm Meeting "MUTATOR GENES THAT PREVENT MICROSATELLITE INSTABILITY IN C. ELEGANS"

J Pothof, R Plasterk, M Tijsterman, K Brouwer

[European Worm Meeting, 2004]

Proper functioning of DNA repair and signaling pathways that maintain the stability of the genome is essential in the prevention of cancer. To identify genes contributing to genomic stability, a frameshift reporter was developed in our lab, which was subsequently used in a genome-wide RNAi screen. This led to the identification of 61 genes that prevent an increase in genomic instability (1). Thirteen genes were shown to be specifically involved in maintaining the stability of microsatellites (short sequence repeats). Among these genes are the four genes known to be involved in the mismatch repair pathway and that are linked to the human cancer syndrome HNPCC (hereditary non-polyposis colon cancer). For three of the yet uncharacterized genes (Y76A2B.5, K09B11.2 and rev-1) we have isolated null alleles. We are currently working on the characterization of these genes by performing epistasis experiments with the mismatch repair pathway and analyzing the mutator phenotypes of the mutants in further detail. 1 Pothof J, van Haaften G, Thijssen K, Kamath RS, Fraser AG, Ahringer J, Plasterk RH, Tijsterman M., Identification of genes that protect the C. elegans genome against mutations by genome-wide RNAi. Genes Dev. 2003 Feb 15;17(4):443-8.

Lemus Vergara, Tzitziki et al. (2019) International Worm Meeting "Genetics of chromosome-X nondisjunction rate variation in C. elegans."

Lemus Vergara, Tzitziki, Kruglyak, Leonid

[International Worm Meeting, 2019]

When cells divide each daughter cell gets a chromosome set. However, the process is imperfect and occasionally chromosomes fail to separate, generating a nondisjunction event. Nondisjunction events can lead to aneuploidy and genetic instability, the former a hallmark of cancer in somatic cells. If nondisjunction occurs during gametogenesis, and the aneuploid gametes become part of a zygote, the resulting embryo has a high probability to abort or to develop physical or mental abnormalities such as human chromosome-21 trisomy, and Turner syndrome, in which females have only one X chromosome. Studying nondisjunction can give us a better understanding of how nondisjunction events occur, and could allow us to predict and alleviate some of its consequences. Our work aims to understand how chromosome nondisjunction is regulated, specifically, how genetic variation and stress influences the rates of chromosome-X nondisjunction. We leverage C. elegans sex determination system to study nondisjunction. Nondisjunction can produce chromosome X-depleted gametes, which upon fertilization form single-X embryos that will give rise to males. Thus, starting with a hermaphrodite population, the percentage of males over one generation is a proxy of chromosome-X nondisjunction rates. We can also increase the proportion of male progeny by subjecting hermaphrodites to stress. To map genetic variants affecting the rate of chromosome-X nondisjunction, we are using a panel of 359 Recombinant Inbred Lines (RILs). This RIL panel was built by mating the reference strain N2 with the CB4856 strain. N2 and CB4856 have different rates of chromosome-X nondisjunction. Thus far, we have phenotyped 90 strains. Preliminary analyses show that the trait is heritable, with genetic variation explaining 46% of the trait variance. An initial QTL mapping revealed that variants on chromosomes II and X might influence chromosome-X nondisjunction. We are currently testing a couple of candidate genes for the increase in chromosome-X nondisjunction.

Yang, Bing et al. (2015) International Worm Meeting "Identifying genes involved in H3K9me2 regulation in C. elegans."

Xu, Xia, Maine, Eleanor, Yang, Bing

[International Worm Meeting, 2015]

Histones can receive many different modifications, and the presence/absence of certain modifications is implicated in transcriptional control and chromosomal events. One modification, H3K9me2, has been found to be associated with unpaired chromatin during meiosis in different organisms, including C. elegans [1-3]. For example, in the male C. elelgans germline, the single X chromosome is unpaired during pachytene and is detected as a single strong focus of H3K9me2 by immuno-labeling. This interesting phenomenon led to the question of how H3K9me2 marks are specifically targeted to their genomic sites.In order to address this question, we are identifying H3K9me2 methyltransferase (MET-2) co-factors. To do so, we generated antibody against MET-2 and epitope-tagged met-2 transgenes. Using these reagents, we performed immunoprecipitation and tandem MS sequencing to recover potential co-factors. Currently, we are testing a list of potential interactors by two different approaches. First, we utilize RNAi to knock down or a mutation to knock out the candidate gene product and then examine the H3K9me2 distribution pattern in the male germline. Second, we perform reverse co-immunoprecipitation by using antibody against the candidate gene product or epitope-tagged transgene product generated either by Mos1-mediated single copy insertion [4] or CRISPR-Cas9 [5] methods. Among the list of candidate genes that have been tested so far, we have identified several genes whose activity impacts H3K9me2 deposition.References: 1. Shiu, P.K., et al., Cell, 2001. 107(7): 905-16. 2. Kelly, W.G., et al., Development, 2002. 129(2): 479-92. 3. Turner, J.M., et al., Nat Genet, 2005. 37(1): 41-7. 4. Frokjaer-Jensen, C., et al., Nat Meth, 2014. 11(5): 529-534. 5. Ran, F.A., et al., Cell. 154(6): p. 1380-1389.