Srinivasan P et al. (2021) Adv Biol (Weinh) "An Autonomous Molecular Bioluminescent Reporter (AMBER) for Voltage Imaging in Freely Moving Animals."

Rothman JH, Theogarajan L, Griffin NM, Thakur D, Saeidi M, Joshi P, Eisdorfer JT, McCotter S, Montell C, Jain A, Kulkarni P, Nguyen-Le A, Srinivasan P

[Adv Biol (Weinh), 2021]

Genetically encoded reporters have greatly increased our understanding of biology. While fluorescent reporters have been widely used, photostability and phototoxicity have hindered their use in long-term experiments. Bioluminescence overcomes some of these challenges but requires the addition of an exogenous luciferin limiting its use. Using a modular approach, Autonomous Molecular BioluminEscent Reporter (AMBER), an indicator of membrane potential is engineered. Unlike other bioluminescent systems, AMBER is a voltage-gated luciferase coupling the functionalities of the Ciona voltage-sensing domain (VSD) and bacterial luciferase, luxAB. When co-expressed with the luciferin-producing genes, AMBER reversibly switches the bioluminescent intensity as a function of membrane potential. Using biophysical and biochemical methods, it is shown that AMBER switches its enzymatic activity from an OFF to an ON state as a function of the membrane potential. Upon depolarization, AMBER switches from a low to a high enzymatic activity state, showing a several-fold increase in the bioluminescence output (L/L). AMBER in the pharyngeal muscles and mechanosensory touch neurons of Caenorhabditis elegans is expressed. Using the compressed sensing approach, the electropharingeogram of the C. elegans pharynx is reconstructed, validating the sensor in vivo. Thus, AMBER represents the first fully genetically encoded bioluminescent reporter without requiring exogenous luciferin addition.

Dance A (2017) Biotechniques "PERSISTENT EPIGENETIC MEMORY."

Dance A

[Biotechniques, 2017]

Nematodes transmit environmental information, such as temperature or food availability, across generations. Amber Dance looks at how this is possible.

Hodgkin JA (1985) Genetics "Novel nematode amber suppressors."

Hodgkin JA

[Genetics, 1985]

Nine amber suppressor mutations were isolated in the nematode Caenorhabditis elegans by reverting amber alleles of a sex-determining gene, tra-3. One suppressor maps to a known locus, sup-5 III , but the other eight map to three new loci, sup-21 X (five alleles), sup-22 IV (two alleles) and sup-23 IV (one allele). Amber alleles of tra-3 and of a dumpy gene, dpy-20, were used to measure the efficiency of suppression; the sup-21 and the sup-22 alleles were both shown to be heterogeneous and generally weaker suppressors than sup-5 alleles, which are homogeneous. The spectrum of mutations suppressed by a strong sup-21 allele, e1957, was investigated and compared to the spectra for the amber suppressors sup-5 III and sup-7 X, using amber alleles in 13 assorted genes. Some of the differences between these spectra may be due to limited tissue specificity in sup-21 expression.-Suppression of dpy-20 was used to show that the sex-linked suppressors sup-7 and sup-21 are not dosage compensated in male (XO) relative to hermaphrodite (XX).-Several uses of amber suppressors are critically discussed: for identifying null mutations, for varying levels of gene activity and for detecting maternal mRNA.

Kimble JE et al. (1982) Nature "Suppression of an amber mutation by microinjection of suppressor tRNA in C. elegans."

Smith TL, Smith JD, Kimble JE, Hodgkin JA

[Nature, 1982]

Informational suppression by nonsense suppressor tRNAs has classically been a powerful tool for study of the mechanism of protein synthesis, to obtain conditional mutants and to demonstrate that a gene encodes a given protein product. In the nematode Caenorhabditis elegans, two genetically identified suppressors, sup-5 and sup-7, have recently been shown to be amber suppressor tRNAs. We report here the microinjection of sup-7 tRNA into the gonad of an animal bearing an amber allele of a maternal-effect mutant affecting sex determination (tra-3). We observe phenotypic suppression in the injected parent's offspring. tRNA from wild-type animals does not show this in vivo suppressor activity, and sup-7 tRNA does not cause suppression of a non-amber allele of the same gene. In vivo suppression of an amber mutant by microinjection provides a new means of gene manipulation in C. elegans.

Fire A et al. (1990) Nucleic Acids Res "Vectors for low copy transformation of C. elegans."

Kondo K, Waterston RH, Fire A

[Nucleic Acids Res, 1990]

The amber suppressor gene sup-7 has been used as a selectable marker to introduce cloned DNA at low copy number into C. elegans chromosomes. We describe structure and several characteristics of seven new sup-7 containing transformation vectors.

Kondo K et al. (1990) J Mol Biol "Genetic and molecular analysis of eight tRNA(Trp) amber suppressors in Caenorhabditis elegans."

Waterston RH, Kondo K, Hodgkin JA, Makovec B

[J Mol Biol, 1990]

Over 100 revertants of five different amber mutants were analyzed by Southern blot hybridization using synthetic oligomers as probes to detect a single base change at the anticodon, CCA to CTA (amber), of tRNA(Trp) genes of Caenohrabditis elegans. Of the 12 members of the tRNA(Trp) gene family, a total of eight were converted to amber suppressor alleles. All eight encode identical tRNAs; three of these are new tRNA(Trp) suppressors, sup-21, sup-33 and sup-34. Previous results had suggested that individual suppressor tRNA genes were expressed differentially in a cell-type- or developmental stage- specific manner. To extend these observations to the new genes and to test the specificity of expression against additional genes, cross suppression tests of these eight amber suppressors were carried out against amber mutations in several different genes including genes likely to be expressed in the same cell-type: three nervous system- affecting genes, two muscle structure-affecting genes and two genes presumed to be expressed in hypodermis. Seven out of eight suppressors could be distinguished one from another by the spectrum of their suppression efficiencies. These results also provide further evidence of cell-type-specific patterns of expression in the nervous system, muscle and hypodermis. The suppression pattern of the suppressor against the two muscle-affecting genes, unc-15 and unc-52, suggested that either the suppressors are expressed in a developmental stage-specific manner or that the unc-52 products are expressed in cell-types other than muscle, possibly hypodermis.

Wills N et al. (1983) Cell "The genes sup-7 X and sup-5 III of C. elegans suppress amber nonsense mutations via altered transfer RNA."

Waterston RH, Wills N, Barnett L, Gesteland RF, Karn J, Bolten SL

[Cell, 1983]

The sup-5 III and sup-7 X suppressors in C. elegans have previously been shown to have many genetic properties in common with tRNA nonsense suppressors of microorganisms. We report here the results of two lines of investigation into the molecular basis of these suppressors. In one, which sought to determine the nature of suppressible alleles, we demonstrate through DNA sequencing studies that a suppressible allele, unc-54(e 1300) I, of the myosin heavy chain gene contains a C leads to T substitution, which changes a glutamine codon to amber terminator at residue 1903. In the other approach, which sought to define the nature of the suppressing activity, we show through in vitro translation studies that tRNA fractions from the suppressor strains, but not wild-type, promote the specific readthrough of amber terminators of three different messenger RNAs. We conclude that sup-5 and sup-7 result in readthrough of amber terminators in vivo through an altered

Parrish AR et al. (2012) ACS Chem Biol "Expanding the genetic code of Caenorhabditis elegans using bacterial aminoacyl-tRNA synthetase/tRNA pairs."

She X, Shen Z, Coin I, Wang L, Dillin A, Briggs SP, Parrish AR, Xiang Z

[ACS Chem Biol, 2012]

The genetic code specifies 20 common amino acids and is largely preserved in both single and multicellular organisms. Unnatural amino acids (Uaas) have been genetically incorporated into proteins by using engineered orthogonal tRNA/aminoacyl-tRNA synthetase (RS) pairs, enabling new research capabilities and precision inaccessible with common amino acids. We show here that Escherichia coli tyrosyl and leucyl amber suppressor tRNA/RS pairs can be evolved to incorporate different Uaas in response to the amber stop codon UAG into various proteins in Caenorhabditis elegans. To accurately report Uaa incorporation in worms, we found that it is crucial to integrate the UAG-containing reporter gene into the genome rather than to express it on an extrachromosomal array from which variable expression can lead to reporter activation independent of the amber-suppressing tRNA/RS. Synthesizing a Uaa in a dipeptide drives Uaa uptake and bioavailability. Uaa incorporation has dosage, temporal, tRNA copy, and temperature dependencies similar to those of endogenous amber suppression. Uaa incorporation efficiency was improved by impairing the nonsense-mediated mRNA decay pathway through knockdown of smg-1. We have generated stable transgenic worms capable of genetically encoding Uaas, enabling Uaa exploitation to address complex biological problems within a metazoan. We anticipate our strategies will be generally extendable to other multicellular organisms.

Fire A (1986) EMBO J "Integrative transformation of Caenorhabditis elegans."

Fire A

[EMBO J, 1986]

A technique for introducing exogenous DNA into the chromosomes of the nematode Caenorhabditis elegans is presented. A cloned C. elegans amber suppressor tRNA gene, sup-7, is used as a selectable marker. The activity of this amber suppressor is selected for by injecting worms which carry an amber termination mutation in a gene (tra-3) whose function is required for fertility. Transient expression of sup-7 is evidenced by the presence of fertile (rescued) animals in the generation after injection. In a fraction of cases, these fertile animals give rise to stable suppressor lines (eight have been characterized so far). Each of the stable suppressor lines carries injected DNA sequences. The suppressor activities have been mapped to chromosomal loci, indicating that the exogenous DNA has integrated into the genome. This technique has been used to introduce a chimeric gene containing a Drosophila heat shock promoter element fused to coding sequences from the Escherichia coli beta-galactosidase gene. This chimeric gene functions and is heat inducible in the resulting stably transformed lines.

Wang X et al. (2020) Genes Nutr "Effects of excess sugars and lipids on the growth and development of Caenorhabditis elegans."

Wang W, Wang J, Zhang L, Wei S, Wang X, Zhang Y, Che H

[Genes Nutr, 2020]

Background: . Methods: Results: . Conclusion: High-sugar and high-lipid intake altered the expression of genes involved in biofilm synthesis, genes that catalyze the synthesis and degradation of endogenous substances, and genes involved in innate immunity, resulting in physiological damage. Furthermore, we explored the protective effect of resveratrol on high-sugar and high-lipid damage to nematodes. Resveratrol plays a role in repairing by participating in the metabolism of foreign substances and reducing cellular oxidative stress.