Jefferson RA et al. (1984) Worm Breeder's Gazette "expression of a chimeric gene transformed into the worm."

Jefferson RA, Hirsh DI

[Worm Breeder's Gazette, 1984]

We have constructed fusions of the col-1 gene from the worm with the E. coli -glucuronidase gene, introduced them into the germline of the worm, and studied their expression. The fusions consist of the promoter region and the first several nucleotides of the col-1 coding sequence, the entire coding sequence of the bacterial -glucuronidase gene, col-1 sequences encoding its 3' intron and the terminator region. The fusions were either in frame or out of frame with the col-1 initiator codon. These constructions were injected into the gonad of gus-1(DH408) worms and detected in the progeny by dot blot hybridization. DH408 is a strain with no detectable -glucuronidase activity, and is used to increase the sensitivity with which we can measure the chimeric activity (Bazzicallupo, Jefferson and Hirsh, unpubl.). -glucuronidase activity was measured in extracts of the transformed worms. DH408 transformed with the in-frame fusion showed glucuronidase activity, while two independent transformed lines carrying the out-of-frame fusion showed no detectable activity. As the DNA was in the long extrachromosomal tandem array, we could use its characteristic instability to perform a cosegregation analysis. There was always a strict correlation between the presence of the transforming DNA and the glucuronidase activity. We verified that the glucuronidase activity in extracts from the transformed line was due to the bacterial enzyme by using affinity purified antibodies directed against the purified bacterial glucuronidase. col-1 has been shown to be transcribed primarily in the embryo ( presumably during the synthesis of the L1 cuticle) but also at lower levels in later stages (Kramer, Cox and Hirsh, in press). To see if the col-1: glucuronidase fusion was being regulated temporally, we prepared extracts from staged populations of transformed worms and measured glucuronidase specific activity. Specific activity is highest in eggs, and decreases markedly in later stages. These experiments are being repeated, but although they are consistent with the wild-type col-1 transcription profiles, they are not sufficient to indicate temporal regulation. To analyze spatial distribution of the glucuronidase activity, we have used enzyme histochemistry and immuno- cytochemical techniques. Although we can visualize activity in eggs using the histochemical stains, we have not yet achieved the resolution to say anything about localization. These experiments are in progress.

Jefferson RA et al. (1981) International C. elegans Meeting "developmental regulation of beta-glucuronidase."

Jefferson RA, Bazzicalupo P, Hirsh DI

[International C. elegans Meeting, 1981]

Jefferson RA et al. (1985) International C. elegans Meeting "expression of chimeric genes in C elegans."

Hirsh DI, Jefferson RA, Burgess SM, Wolf N

[International C. elegans Meeting, 1985]

Jefferson RA et al. (1983) International C. elegans Meeting "plasmid expression vectors for the in vivo analysis of regulatory sequences."

Jefferson RA, Hirsh DI

[International C. elegans Meeting, 1983]

Jefferson RA et al. (1987) J Mol Biol "Expression of chimeric genes in Caenorhabditis elegans."

Klass MR, Hirsh DI, Wolf N, Jefferson RA

[J Mol Biol, 1987]

We have shown the expression of transformed genes in the nematode Caenorhabditis elegans using a new gene fusion system. Vectors consisting of the flanking regions of a collagen gene (col-1) or a major sperm protein gene of C. elegans fused to the Escherichia coli uidA gene, encoding beta-glucuronidase, were microinjected into worms and found to be propagated as high-copy extrachromosomal tandem arrays. We have detected beta-glucuronidase activity in transformed lines, and have shown that the activity is dependent upon the correct reading frame of the construction and on the presence of the worm sequences. The enzyme activity was shown to be encoded by the chimeric beta-glucuronidase gene by co-segregation analysis and by inactivation with specific antisera. Expression is at a very low level, and seems to be constitutive. We have used histochemical techniques to visualize the enzyme activity in embryos.

Liao Y et al. (1999) J Biol Chem "RA-GEF, a novel Rap1A guanine nucleotide exchange factor containing a Ras/Rap1A-associating domain, is conserved between nematode and humans."

Liao Y, Goshima M, Kariya K-i, Hu CD, Kataoka T, Jin TG, Watari Y, Yamawaki-Kataoka Y, Okada T, Gao X, Shibatohge M

[J Biol Chem, 1999]

A yeast two-hybrid screening for Ras-binding proteins in nematode Caenorhabditis elegans has identified a guanine nucleotide exchange factor (GEF) containing a Ras/Rap1A-associating (RA) domain, termed Ce-RA-GEF. Both Ce-RA-GEF and its human counterpart Hs-RA-GEF possessed a PSD-95/DlgA/ZO-1 (PDZ) domain and a Ras exchanger motif (REM) domain in addition to the RA and GEF domains. They also contained a region homologous to a cyclic nucleotide monophosphate-binding domain, which turned out to be incapable of binding cAMP or cGMP. Although the REM and GEF domains are conserved with other GEFs acting on Ras family small GTP-binding proteins, the RA and PDZ domains are unseen in any of them. Hs-RA-GEF exhibited not only a GTP-dependent binding activity to Rap1A at its RA domain but also an activity to stimulate GDP/GTP exchange of Rap1A both in vitro and in vivo at the segment containing its REM and GEF domains. However, it did not exhibit any binding or GEF activity toward Ras. On the other hand, Ce-RA-GEF associated with and stimulated GDP/GTP exchange of both Ras and Rap1A. These results indicate that Ce-RA-GEF and Hs-RA-GEF define a novel class of Rap1A GEF molecules, which are conserved through evolution.

Lin C et al. (2019) Biofactors "Rosmarinic acid improved antioxidant properties and healthspan via the IIS and MAPK pathways in Caenorhabditis elegans."

Xi Y, Lin C, Zhong Q, Zhang X, Zheng H, Xiao J, Cao Y, Chen Y

[Biofactors, 2019]

Rosmarinic acid (RA) has a wide range of biological effects, including the antioxidation and antiaging. However, the detailed mechanisms remain unclear but highly attractive. Herein, RA promoted lifespan and motoricity in a dose-dependent manner, and reduced fat store without threatening fertility in Caenorhabditis elegans. In term of antioxidant efficacy, catalase activity, glutathione peroxidas activity, reduced glutathione content, and reduced glutathione/oxidized glutathione ratio were enhanced. And malondialdehyde content was diminished significantly. Moreover, RA increased survival under acute oxidative and thermal stress, and suppressed intestinal lipofuscin accumulation. So the improvement of lifespan mediated by RA could be related with its strong antioxidant properties. Furthermore, RA was absorbed by worms. Further research in pursuit of the mechanism showed that longevity induced by RA was involved with the genes sod-3, sod-5, ctl-1, daf-16, ins-18, skn-1, and sek-1, but was independent of subcellular localization of DAF-16. These findings indicated that RA had a potential for promoting healthy lifespan.

Chen Y et al. (2024) Mol Neurobiol "Rosmarinic acid ameliorated oxidative stress, neuronal injuries, and mitochondrial dysfunctions mediated by polyglutamine and ɑ-synuclein in Caenorhabditis elegans models."

Zeng Y, Liu Y, Liu Q, Cao Y, Xu R, Chen W, Liu G, Chen Y

[Mol Neurobiol, 2024]

Numerous natural antioxidants have been developed into agents for neurodegenerative diseases (NDs) treatment. Rosmarinic acid (RA), an excellent antioxidant, exhibits neuroprotective activity, but its anti-NDs efficacy remains puzzling. Here, Caenorhabditis elegans models were employed to systematically reveal RA-mediated mechanisms in delaying NDs from diverse facets, including oxidative stress, the homeostasis of neural and protein, and mitochondrial disorders. Firstly, RA significantly inhibited reactive oxygen species accumulation, reduced peroxide malonaldehyde production, and strengthened the antioxidant defense system via increasing superoxide dismutase activity. Besides, RA reduced neuronal loss and ameliorated polyglutamine and ɑ-synuclein-mediated dyskinesia in NDs models. Further, in combination with the data and molecular docking results, RA may bind specifically to Huntington protein and ɑ-synuclein to prevent toxic protein aggregation and thus enhance proteostasis. Finally, RA ameliorated mitochondrial dysfunction including increasing adenosine triphosphate and mitochondrial membrane potential levels and rescuing mitochondrial membrane proteins' expressions and mitochondrial structural abnormalities via regulating mitochondrial dynamics genes and improving the mitochondrial kinetic homeostasis. Thus, this study systematically revealed the RA-mediated neuroprotective mechanism and promoted RA as a promising nutritional intervention strategy to prevent NDs.

Wong, Wan-Rong et al. (2021) MicroPubl Biol

Oh, Jun Young, Wong, Wan-Rong, Maher, Shayda, Sternberg, Paul W, Gharib, Shahla, Brugman, Katherine I

[MicroPubl Biol, 2021]

Retinoic acid (RA), the active metabolite of vitamin A, broadly regulates gene expression. The RA signaling pathway plays an essential role in embryonic development, including the development of the body axis, eye, brain, and heart (Ghyselinck & Duester, 2019). One of the key enzymes in the biosynthesis of RA is aldehyde dehydrogenase 1 family member A3 (ALDH1A3). ALDH1A3 converts retinaldehyde to retinoic acid and it is expressed early in forebrain development (McCaffery & Drager, 1994). Several mutations in ALDH1A3 have been implicated in patients with autosomal recessive microphthalmia and other neurological disorders (Fares-Taie et al., 2013; Roos et al., 2014). However, current animal models of ALDH1A3 have large truncations of the protein. Direct evidence of the effects of missense variants on ALDH1A3 protein activity has not yet been obtained.

Hirsh DI et al. (1985) Cold Spring Harb Symp Quant Biol "Genes affecting early development in Caenorhabditis elegans."

Jefferson RA, Kemphues KJ, Stinchcomb DT, Hirsh DI

[Cold Spring Harb Symp Quant Biol, 1985]

We have identified several genes that appear to control the early events in the development of Caenorhabditis elegans. We continue to study them genetically and characterize them morphologically. We describe here three of these genes that appear to be involved in establishing the organization of the embryonic cytoplasm and in positioning the early cleavage furrows. In addition, we review our continuing studies on the characterization of the C. elegans genome, the goal of which has been to facilitate the study of the molecular basis of the role of these genes in early development...