Babbar R et al. (1998) European Worm Meeting "Analysis of the regulatory factors involved in cdh-3 and cdh-9 expression"

Pettitt J, Babbar R

[European Worm Meeting, 1998]

To date, 10 members of the cadherin superfamily have been identified in C. elegans (see abstract by Mondragon and Pettitt). We are interested in understanding the regulation of cadherin expression and to what extent the regulatory mechanisms are conserved between phyla. For reasons outlined below, we are beginning this work by focusing upon the regulation of two cadherin genes, cdh-3 and cdh-9. The upstream region of the cdh-3 gene has been implicated in producing a phenotype termed "Pun" (Pharynx unattatched) when it is present in high copy number transgenes. This phenotype is attributable to the fact that in these animals the connection between the pharynx and the epidermis surrounding the buccal opening fails to from properly and therefore the nematode has effectively no "mouth". The phenotype is lethal, as the nematode is unable to feed. cdh-3 is expressed in the cells responsible for the connection between the pharynx and the epidermis and it seems likely that the Pun phenotype is due to the titration of a trans-acting factor, or factors, by the transgenic cdh-3 promoter sequences. This may provide a means to identify the factor(s) involved. We are attempting to identify the region of the cdh-3 promoter involved by making deletion derivatives of a gfp-based promoter construct and assaying both their expression patterns and whether they confer a Pun phenotype. The C. briggsae orthologue of cdh-9 has been sequenced and the exon structure of the two genes is highly conserved. In addition, regions upstream of the start site and within the first intron are conserved, suggesting that these may represent regulatory elements. The initial goal of this project is to establish the expression pattern of cdh-9, firstly in C. elegans and then in C. briggsae. This will be done by tagging the cdh-9 gene with gfp in a construct and assaying expression in both nematodes. The C. briggsae orthologue will be similarly tagged and analysed. These experiments will have two objectives. Firstly, to confirm the observations made in silico. Secondly, to determine whether any divergence in the expression patterns of this gene in the two nematodes exists, and if so whether this correlates with differences in the cis-acting regulatory sequences.

Pettitt J et al. (2000) European Worm Meeting "Regulation of cdh-3 and cdh-9 expression"

Babbar R, Pettitt J

[European Worm Meeting, 2000]

Cadherins are transmembrane glycoproteins that mediate calcium dependent homophilic cell-cell adhesion. Thirteen cadherins have been identified in C. elegans of which 12 are protocadherins. We are interested in understanding how protocadherin genes are transcriptionally regulated in the development of C. elegans. We are concentrating on cdh-3 and cdh-9 as both the C. briggsae orthologues have been sequenced. Sequence comparisons of cdh-9 in C. elegans and its orthologue in C. briggsae show that the gene is highly conserved, this is also the case for cdh-3 in C. elegans and its respective orthologue.In addition, there are regions upstream of the first exon that are conserved, suggesting that these may represent regulatory elements. The expression patterns of both genes and their respective orthologues will be established in C. elegans and C. briggsae. From this we will be able to determine if there is any divergence in the expression patterns of the genes in the two nematodes; if so, this may correlate with differences in cis-acting regulatory sequences. The upstream region of cdh-3 has been implicated in producing a phenotype termed Pun (Pharynx unattached) when present as a transgene at high copy number. The phenotype is attributable to the fact that the connection between the pharynx and the buccal opening fails to form properly, therefore the nematode effectively has no mouth. The phenotype is lethal as the nematode is unable to feed. As cdh-3 is expressed in the cells responsible for the connection between the pharynx and the epidermis, the Pun phenotype is probably due to the titration of a trans-acting factor, or factors, by the transgenic cdh-3 promoter sequences. This may provide a means to identify the factor(s) involved. We are attempting to identify the region of the cdh-3 promoter involved by making deletion derivatives of a gfp-based promoter construct, and assaying their expression patterns in combination with the ability to induce the Pun phenotype. So far, we have identified a promoter region required for embryonic expression of cdh-3. Transgenes missing this region fail to express gfp in the embryo. Consistent with this, we do not see any Pun phenotypes in animals transgenic for this promoter deletion. Sequence comparison has also shown that there is a high degree of identity between the 800 bp region preceding the ATG codon of cdh-3 in C. elegans, and the equivalent region in C. briggsae. This indicates that a putative transcription site may be located in this region. We are currently attempting to refine the location of the putative promoter element. For cdh-9 we are using a PCR based approach to tag the cdh-9 promoter region with gfp. This will be used to establish an expression pattern in both C. elegans and C. briggsae. We also plan to use the same approach to tag the C. briggsae orthologue and analyse it in C. elegans and C. briggsae.

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.

Birsoy B et al. (2010) Biology of the C. elegans Male, Madison, WI "Novel Left-Right Asymmetries of Male C.elegans"

Rothman J H, Choi S, Birsoy B, Downes J C, Bloss T A

[Biology of the C. elegans Male, Madison, WI, 2010]

While the mechanism that breaks left-right (L-R) anatomical asymmetry has been described in C. elegans, we have found that at least one additional mechanism must exist to generate L-R differences in the deployment of alternative cell death pathways in the male tail, male mating behavior and patterning of the male gonad. Upon recognizing a hermaphrodite, males initiate backward locomotion and continuously scan along the hermaphrodite's body. When their tails reach either the head or tail of the hermaphrodite, males turn to maintain contact and then continue backward locomotion on the opposite side of the hermaphrodite. We found that this turning behavior shows a distinct right-handed bias: when individual males were allowed to mate with lin-2(e1309) vulvaless hermaphrodites, >70% of the turns when made over the hermaphrodites' body (away from the agar surface) and >55% of turns when made under the hermaphrodites' body (toward the agar surface) are right-handed. To determine whether this bias in turning direction correlated with L-R asymmetry in the male mating structure, which results from stochastic EGL-1-dependent loss of sensory rays, we examined the turning bias in egl-1(n1084n3082) mutants. Wild-type males lack rays more frequently on the right and egl-1 mutant males have no ray loss but still display the right-hand turning bias. To assess whether this L-R behavioral bias is dependent on anatomical asymmetry, we analyzed gpa-16(it143) mutants in which the L-R asymmetry of the internal organs is reversed as a result of the reversal in the early embryonic symmetry break. Males with reversed anatomical asymmetry showed a virtually identical right hand bias in turning, demonstrating that an asymmetry-determining system that is independent of the previously described L-R symmetry breaking event must control this behavior. During the characterization of the gonad reversals of males, we also observed a previously unreported temperature sensitive reversal of L-R asymmetry of the male gonads in N2 (Bristol) and a Hawaiian wild isolate. We will describe our recent findings on these novel L-R asymmetries of the C.elegans male anatomy and behavior.

L C Bowen et al. (2002) European Worm Meeting "The C. elegans cyclic AMP-dependent protein kinase (PK-A) catalytic subunit gene (kin-1)"

L C Bowen, M Tabish, M J Fisher, H H Rees, R A Clegg

[European Worm Meeting, 2002]

PK-A is one of the major multifunctional ser/thr protein kinases found in eukaryotic organisms. The holoenzyme is comprised of two catalytic (C) and two regulatory (R) subunits. Both R and C subunits occur in multiple isoforms encoded by distinct genes. In many organisms, the genes that encode the C subunits also show alternative splicing behaviour. This generates an even broader array of C subunit heterogeneity. The diversity of C subunit polypeptides may enable this protein kinase to selectively target substrate polypeptides for phosphorylation.

Martyna W Pastok et al. (2007) International Worm Meeting "Studies on the C. elegans Protein Kinase A (PK-A) Catalytic and Regulatory Subunit Genes."

Huw H Rees, Martyna W Pastok, Michael J Fisher, Caroline Dart, Patrica A Murray

[International Worm Meeting, 2007]

The cyclic AMP-dependent protein kinase (protein kinase A; PK-A) holoenzyme consists of two catalytic (C) subunits bound to two regulatory (R) subunits, forming an inactive heterotetramer (R2C2). Binding of cyclic AMP to the R subunits results in the dissociation of catalytically active monomeric C subunits. These C subunits catalyse the protein phosphorylation events underlying the diverse range of cellular processes triggered by alterations in cyclic AMP concentration. In C. elegans, C and R subunits are encoded by the kin-1 and kin-2 genes respectively. We have undertaken both bioinformatic and molecular studies on the structure and expression of these genes, in C. elegans. We have also undertaken comparative bioinformatic studies on orthologus genes, and gene products, in C. briggsae and C. remanei. These studies have highlighted the potential significance of alternative splicing processes, as a means of generating multiple isoforms of both the C and R subunits in C. elegans. The possible significance of such multiple isoforms is discussed in the context of the subcellular distribution of PK-A and in relation to the targeting of specific phosphorylation events, in responses to changes in intracellular cyclic AMP concentration.