Feichtinger R et al. (1993) International C. elegans Meeting "Maternal effect lethals on LG IV and V."

Schnabel R, Feichtinger R, Schnabel H

[International C. elegans Meeting, 1993]

Feichtinger R et al. (1995) International C. elegans Meeting "MATERNAL SCREEN ON LG IV AND V FINISHED"

Schnabel R, Weigner C, Feichtinger R, Schnabel H

[International C. elegans Meeting, 1995]

As part of our screens for maternal effect embryonic lethal mutations we screened on chromosomes IV and V with nT1, which balances about 30% of the genome according to cDNA count. From 14,700 EMS mutagenized strains we isolated 390 mutations. They were mapped to deficiencies and fall into about 150 complementation groups. For about 50 genes we isolated more than one allele. The distribution of the number of alleles is very disperse, the most frequent gene gave 35 alleles. A thorough statistical analysis (excluding the singly hit genes) allows us to estimate the total number of maternal genes for the genome to be about 400. Using a bootstrap method we determined the accuracy of this estimation to be 250 - 800 genes within one standard deviation. Of the previously known maternal genes in the region we have tested skn-1, apx-1 and par-1 for complementation and found 0, 7 and 2 new alleles, respectively. The gene with 35 alleles seems to be a new one, located under sDf35 to the right of unc-42. Strong alleles show no, or almost no, 3NB12 pharyngeal muscle staining. They show lineage aberrations mostly in the AB and MS lineages. The preliminary TSP period is most severe from the 4-cell to the 16-cell-stage. The phenotypic characterization of the remaining 149 genes is in progress . . . . . . . . .

Feichtinger R et al. (1991) International C. elegans Meeting "A SCREEN FOR MATERNAL EFFECT LETHALS USING THE BALANCER nTl"

Schnabel R, Schnabel H, Feichtinger R

[International C. elegans Meeting, 1991]

In the first 120 minutes of development of C. elegans, until the embryo consists of 28 cells, a great deal of determination is already achieved: the embryonic axes have been established, all embryonic founder cells have been generated, the monoclonally derived germline and gut have been segregated and the primary inductive events have already occurred. Most of the early decisions are thought to be maternally controlled. We are screening for maternal effect lethal mutations with the aim to saturate part of the genome. The balancer nTl is used in order to maintain nonconditional mutants as heterozygotes. With nTl, 60% of chromosome IV and 65% of chromosome V, according to the physical map, are balanced, accounting for V5 of the genome. We want to complement the mutations prior to phenotypic analysis in order to look at several independently isolated mutant alleles for each locus. Since complementation analysis raises in effort quadratically with the number of mutations, grouping by deficiency-mapping is very profitable. In our first set we obtained maternal effect lethals at a rate of 0.015 per haploid genome. We intend to isolate about 420 mutations (corresponding to 28000 haploid genomes). Using a statistical analysis of our data of a similar screen with mnCl we can calculate a projection for this screen. A negative binomial probability distribution is used to simulate the more frequently appearing loci. We estimate a mean saturation of 3 and a remarkably large number of 95 loci of this kind, i.e. maternal genes, in this region. Half of them would be represented with more than one mutant allele.

Molin LF et al. (1997) International C. elegans Meeting "PES-1 AN EARLY MARKER AND A TOOL TO STUDY THE COMPLEXITY OF C. ELEGANS EMBRYOGENESIS"

Molin LF, Hope IA, Schnabel H, Schnabel R, Feichtinger R, Kaletta T

[International C. elegans Meeting, 1997]

How many of the 15,000 genes in the C. elegans genome are involved in building the complexity of the AB lineage during early embryogenesis? Since many C. elegans genes are essential and those involved in early embryogenesis are maternally supplied, we focused on a collection of maternal-effect embryonic lethal mutants. The gene pes-1 is expressed in a subset of AB cells during early embryogenesis and is a suitable marker to identify mutants with an altered AB lineage patterning. The pes-1::lacZ expression pattern was thus analysed in mutants for 41 genes randomly chosen from the collection described above. While mutants in 26 genes (63%) demonstrate a wild-type pes-1::lacZ pattern, mutants in 15 genes (37%) demonstrate an altered expression pattern. From screens for maternal-effect embryonic lethal mutations, one can estimate that about 500 maternally supplied genes are essential for embryogenesis. About 50% of these genes are involved in very early processes and were not taken into account in this study. This study thus suggests that about 100 maternally supplied genes are involved in the patterning of the AB lineage during the first 100 minutes after division of the zygote. The pes-1::lacZ expression pattern was also analysed in ablated embryos and in mutants for genes already known to affect AB lineage patterning (glp-1, apx-1, pie-1, skn-1). The pes-1::lacZ expression pattern we observed is consistent with the blastomere transformations proposed to occur in these mutants or ablated embryos. These results support the use of pes-1 as an early marker and as a tool with which to investigate new hypotheses concerning blastomere transformation.

Li-Leger, E et al. (2019) International Worm Meeting "Identification and characterization of maternal-effect genes involved in early embryonic development."

Feichtinger, R., Li-Leger, E, Schnabel, R., Moerman, D. G., Holzkamp, H., Flibotte, S.

[International Worm Meeting, 2019]

Most of early embryonic development is regulated by gene products - RNAs and proteins - that are supplied to the egg by the mother. These maternal-effect gene products regulate critical processes before zygotic gene expression is activated. While much of early development is controlled by maternal-effect genes, we still know very little about the precise role of many of these genes. With the advent of whole genome sequencing (WGS), and as the cost of sequencing drops, large legacy mutant collections are becoming increasingly accessible to molecular analysis. The large array of maternal-effect lethal mutants, many of which are temperature-sensitive, isolated by the Schnabel lab is one such collection. We are analyzing a limited set of mutant strains from this collection with maternal-effect lethal phenotypes, with the aim of identifying novel members in this class of essential genes. These mutations have been isolated and mapped to 98 complementation groups on four different chromosomes, and we are using WGS to identify the genes of interest. Our strategy is to sequence two alleles of each complementation group and to focus on genes mutated in both alleles. To date, 28 of these genes have been identified, including 11 genes for which mutant alleles were not previously available: C34D4.4, cpt-2, D2096.12, dgtr-1, dlat-1, F21D5.1, F56D5.2, nstp-2, T22B11.1, trcs-1, and vars-1. For the novel essential genes uncovered by our analysis, we are documenting the stage of arrest and terminal phenotypes of the lethal embryos using DIC microscopy. This preliminary phenotypic characterization will provide a basis for further studies into gene function. Identifying and characterizing alleles that cause developmental defects in C. elegans will further our understanding of the molecular basis of development and embryogenesis. Furthermore, given that approximately 40% of C. elegans genes have human orthologs and that mutations in essential genes are often implicated in human disease, some of the alleles in this collection are likely to be of interest in various fields of research.

Rand JB et al. (2000) East Coast Worm Meeting "UNC-13 in the C. elegans Nervous System"

Kohn RE, Rand JB, McManus JR, Moulder GL, Duke A, Barstead RJ, Duerr JS

[East Coast Worm Meeting, 2000]

C. elegans unc-13 and its homologues in vertebrates and Drosophila are involved in neurotransmitter release. UNC-13 has several regions homologous to PKC regulatory domains; these domains confer it with calcium, phorbol ester and phospholipid binding properties (Maruyama and Brenner, PNAS 88, 1991). A 5.9kb transcript coding for a 200kDa protein was initially identified (now designated L-R for left and right regions). We have identified two additional types of transcripts. One transcript includes a 1kb novel exon (L-M-R, M for middle region) and another transcript lacks the 5' region included in the other two transcripts (M-R). All three transcripts are identical at the 3' end (R). C. elegans with mutations in the 5' end of the gene (L) alter two types of transcripts (L-R and L-M-R) resulting in an uncoordinated coily phenotype and resistance to the anti-cholinesterease, aldicarb. A 2.7kb deletion near the 3' end (R) (identified by Bob Barstead using PCR analysis) affects all unc-13 transcripts and results in a lethal phenotype. Antibodies recognizing the N-terminal region of UNC-13 (L) label synapses, but not synaptic vesicles, of most or all neurons; many mutations in L and R remove staining with this antibody. Supported by grants from the NIH and OCAST.

Abergel, Z. et al. (2017) International Worm Meeting "Adaptation of the nematode C. elegans to hypoxia and reoxygenation stress reveals an unexpected function of the neuroglobin GLB-5 in innate immunity."

Zuckerman, B., Zelmanovich, V., Abergel, Z., Abergel, R., Gross, E., Smith, Y., Romero, L., Livshits, L.

[International Worm Meeting, 2017]

Deprivation of oxygen (hypoxia) followed by reoxygenation (H/R stress) is a major component in several pathological conditions such as vascular inflammation, myocardial ischemia, and stroke. However how animals adapt and recover from H/R stress remains an open question. Previous studies showed that the neuroglobin GLB-5(Haw) is essential for the fast recovery of the nematode Caenorhabditis elegans (C. elegans) from H/R stress. Here, we characterize the changes in neuronal gene expression during the adaptation of worms to hypoxia and recovery from H/R stress. Our analysis shows that innate immunity genes are differentially expressed during both adaptation to hypoxia and recovery from reoxygenation stress. Moreover, we reveal that the prolyl hydroxylase EGL-9, a known regulator of both adaptation to hypoxia and the innate immune response, inhibits the fast recovery from H/R stress through its activity in the O2-sensing neurons AQR, PQR, and URX. Finally, we show that GLB-5(Haw) acts in AQR, PQR, and URX to increase the tolerance of worms to bacterial pathogenesis. Together, our studies suggest that innate immunity and recovery from H/R stress are regulated by overlapping signaling pathways.

A V Bicknell et al. (2002) European Worm Meeting "The C. elegans F47F2.1 gene encodes a cyclic AMP-dependent protein kinase (PK-A) catalytic subunit"

A V Bicknell, M J Fisher, M Tabish, R A Clegg, H H Rees

[European Worm Meeting, 2002]

PK-A mediates all known effects of cyclic AMP on cellular activity in eukaryotes. The holoenzyme is an inactive tetramer of two regulatory (R) subunits and two catalytic (C) subunits. Following binding of cyclic AMP to the R subunits, dissociation of active C-subunits occurs. In mammals, , and isoforms of C-subunit, encoded by different genes, have been identified.

Angelo RG et al. (1997) International C. elegans Meeting "DISCOVERY OF A POTENTIAL ANCHOR/SCAFFOLD PROTEIN FOR C. ELEGANS PROTEIN KINASE A (PKA)"

Angelo RG, Rubin CS

[International C. elegans Meeting, 1997]

C. elegans PKA is composed exclusively of catalytic and regulatory (R) subunits encoded by the kin-1 and kin-2 genes. Since C. elegans lacks other PKA isoforms, this enzyme must disseminate signals carried by cAMP to all cell compartments. Approximately 60% of total R (PKA) is in the particulate fraction of disrupted C. elegans, indicating that protein/protein interactions may diversify PKA signaling by anchoring the kinase at specific intracellular locations. Co-localization of PKA with upstream activators and/or downstream effectors can create target sites for cAMP action. To understand the mechanism of PKA localization in C. elegans, a cDNA expression library was screened with recombinant radiolabeled-R (0.5 nM) to obtain high-affinity binding proteins. A cDNA encoding a novel, 143 kDa A kinase anchor protein (AKAP1) was retrieved and sequenced. The corresponding gene (rap-1) is located in LGII and contains 17 exons. AKAP1 is an acidic protein (pI=4.4) that is unrelated to previously characterized proteins. C. elegans R is avidly bound by both soluble and immobilized fragments of AKAP1. Competition binding studies indicate that C. elegans R is a preferred ligand, whereas mammalian RII and RI isoforms are only weakly sequestered. Scatchard analysis yielded a Kd value of ~10 nM for the R/AKAP1 complex. Deletion mutagenesis, coupled with protein expression in E. coli and in vitro assays, demonstrated that residues 235 to 255 govern high-affinity binding of R by AKAP1. A hydrophobic surface generated by amino acids with branched aliphatic side chains may be a key determinant of R binding activity. Site directed mutagenesis will pinpoint essential residues involved in PKA binding. Studies aimed at identifying the AKAP1 binding site on R are also in progress. High-affinity anti-AKAP1 IgGs are being used to determine (a) whether AKAP1 expression is developmentally-regulated and cell- specific and (b) the relationship between R (PKA) and AKAP1 in vivo.

Hicks, Tara et al. (2021) International Worm Meeting "R-loop-induced irreparable DNA damage in C. elegans meiosis"

Hicks, Tara, Koury, Emily, Williams, Cameron, Smolikove, Sarit

[International Worm Meeting, 2021]

Most DNA-RNA hybrids are formed naturally during transcription and are composed of a nascent RNA strand hybridized to DNA as part of R-loops. The accumulation of these structures in S-phase can result in replication-transcription conflict, an outcome which can lead to the formation of double strand breaks (DSBs). While R-loops' role in mitotically dividing cells has been characterized, there are only a handful of studies describing the effect of R-loops in meiosis and these studies present a complex picture of the outcome of R-loop formation on germ cells. Here we show that DSBs formed by R-loops trigger an altered cellular response to DNA damage. RNase H is an enzyme responsible for degradation of the RNA strand in DNA-RNA hybrids and plays an essential role in preventing this outcome and its deleterious consequences. Using null mutants for the two Caenorhabditis elegans genes encoding for RNase H1 and RNase H2 (hereby rnh mutants), our studies explore the effects of replication stress-induced DNA-RNA hybrid accumulation on meiosis. As expected, rnh mutants exhibit an increase in R-loop formation. Consequently, an elevation of DSBs in germline nuclei is evidenced by the accumulation of RAD-51 foci. Despite no repair mechanism abrogation, rnh mutants fail to repair all DSBs generated, leading to a fragmentation of chromosomes in diakinesis oocytes. By combining our double mutant with a spo-11 null mutation, we show that although replicative defects are the main contributor to the phenotype, R-loops formed in meiosis are likely contributors as well. We present evidence that while rnh mutants accumulate DNA-RNA hybrids and subsequent DSBs may signal a degree of checkpoint activation in mitosis, some damaged nuclei prevail past the checkpoint, enter into meiosis, and remain unrepaired throughout. Moreover, we find no evidence of an increase in apoptosis, which indicates that DNA damage generated by R-loops remain undetected by an apoptotic checkpoint. This data altogether points to DSBs initiated by R-loops representing an irreparable type of DNA damage that evades cellular machineries designed for damage recognition.