Zimmer M (2009) Chem Soc Rev "GFP: from jellyfish to the Nobel prize and beyond."

Zimmer M

[Chem Soc Rev, 2009]

On December 10, 2008 Osamu Shimomura, Martin Chalfie and Roger Tsien were awarded the Nobel Prize in Chemistry for "the discovery and development of the green fluorescent protein, GFP". The path taken by this jellyfish protein to become one of the most useful tools in modern science and medicine is described. Osamu Shimomura painstakingly isolated GFP from hundreds of thousands of jellyfish, characterized the chromophore and elucidated the mechanism of Aequorean bioluminescence. Martin Chalfie expressed the protein in E. coli and C. elegans, and Roger Tsien developed a palette of fluorescent proteins that could be used in a myriad of applications.

Mendenhall, Alexander et al. (2015) International Worm Meeting "Effects of introns on gene expression."

Mendenhall, Alexander, Brent, Roger, Sands, Bryan

[International Worm Meeting, 2015]

In C. elegans, as in the early days of recombinant DNA in mammalian cells, introns are generally used to increase transgene expression. Specifically, to increase expression, most C. elegans promoter-fused-to-fluorescent-protein reporter genes in use have three synthetic introns developed by the Fire lab inserted into the coding sequence of mCherry or GFP. But exactly how much more expression do those introns result in? Are all three introns necessary for the increased expression? Here, using single copy reporters integrated into the same defined locus, we measured the effects of different numbers, positions and sequences of introns on mean gene expression. We quantified expression from whole animals in flow and individual intestine cells using a point scanning confocal microscope. Preliminary findings suggest that, relative to no intron reporter gene controls, different combinations of the original three synthetic introns can result in increased or decreased mean expression. We intend to use these combinations to set reporter gene expression output to desired levels, for example, so as to be within the dynamic range of measurement of other reporters whose products are expressed in the same cells or in the same visual field. .

Frantz S (2001) Nat Rev Mol Cell Biol "Role model."

Frantz S

[Nat Rev Mol Cell Biol, 2001]

As genetic methods in C. elegans models have been a crucial factor in the identification of several cellular pathways, the authors of both papers hope their findings will provide new insights into the regulation and function of mammalian p53. Another potential key area of research could spring from Brent Derry and colleagues' isolation of a genomic p53 mutation, which could provide the possibility for screening tools.

Mendenhall, Alexander et al. (2015) International Worm Meeting "Reproducible quantification of gene expression in single cells of live adult animals."

Tedesco, Patricia, Brent, Roger, Sands, Bryan, Mendenhall, Alexander, Johnson, Thomas

[International Worm Meeting, 2015]

In multicellular organisms such as Caenorhabditis elegans, differences in complex phenotypes such as lifespan correlate with the level of expression of particular engineered reporter genes. In single celled organisms, quantitative understanding of responses to extracellular signals and of cell-to-cell variation in responses has depended on precise measurement of reporter gene expression. We developed microscope-based methods to quantify reporter gene expression in cells of Caenorhabditis elegans with low measurement error. At the level of cells, in animals with single copy reporters, the pattern of expression in cells within the tissue was highly stereotyped. In animals with multicopy reporters, the cell-specific expression pattern was also stereotyped, but distinct, and somewhat more variable. Our methods are rapid and gentle enough to allow quantification of expression in the same cells of an animal at different times during adult life. We hope they will allow investigators to use changes in reporter expression in single cells in tissues as quantitative phenotypes, and link those to their molecular causes. Moreover, by diminishing measurement error, they should make possible dissection of the causes of the remaining, real, variation in expression. We hope that understanding such variation will help reveal its contribution to differences in complex phenotypic outcomes in multicellular organisms.

Mendenhall, Alexander et al. (2015) International Worm Meeting "Mechanisms of animal-to-animal and cell-to-cell variation in gene expression in adult hermaphrodites."

Mendenhall, Alexander, Brent, Roger, Sands, Bryan, Johnson, Thomas, Tedesco, Patricia

[International Worm Meeting, 2015]

In isogenic populations of C. elegans cultured under homogeneous conditions, differences in the amount of GFP produced under control of the hsp-16.2 promoter predict differences in lifespan. Animals with high expression enjoy longer lifespan, longer healthspan and higher thermotolerance. How do animals end up in this privileged physiological state? We showed previously that variation (here, the coefficient of variation or CV) in hsp-16.2 reporter gene expression was constant among different strains bearing reporter constructs with differences in locus of integration, different copy numbers, and different fluorescent proteins. Here we sought to understand the origins of the variation in expression that results in high expression animals. We used a genetic and cell biological approach to study the origins of variable expression in individual animals and individual cells. Our preliminary results show that, at the level of the whole animal two distinct systems affect variation in gene expression, one of which increases, and the other of which decreases (canalizes) interindividual differences. At the cell level there is not much "intrinsic noise" in reporter expression (that is, fluorescent signal from two different differently colored reporter proteins driven by two different instances of the same promoter is highly correlated). In contrast to some studies in single cell organisms, we find that, when we measure the same cells in different animals, the ratio of fluorescent signal from two different fluorescent proteins driven by two different promoters is almost the same. It is possible that this limitation of variation in gene expression may reflect a need to tightly coordinate expression of different genes during metazoan development and adult life.

Livingstone D (1989) Worm Breeder's Gazette "some more unc-31 sequence."

Livingstone D

[Worm Breeder's Gazette, 1989]

I am continuing the molecular work on the unc-31 gene started by Roger Hoskins. Roger isolated a 17.5kb genomic clone in lambda2001 which he showed was capable of transformation rescue of unc-31 mutants. He also partially sequenced a 4.5kb fragment contained within this clone which covers some of the polymorphisms associated with mutant alleles of the gene. [See Figure 1] I have made subclones of this phage in lambda2001 and have used them in an attempt to narrow down the position of the gene by transformation rescue. A phage containing the 13.5kb XhoI fragment seems to rescue although this result has still to be confirmed. No rescue has been obtained with phage containing the 13.5kb SacI fragment or the 10.5kb SacI-XhoI fragment. I am currently sequencing the 13.5kb rescuing fragment. Several open reading frames have been found which could be spliced together inframe. Some of these have been confirmed by sequencing partial cDNA clones isolated from Julie Ahringers mixed stage cDNA library. The gene covers more than 8kb of genomic DNA. The 5' end is near or beyond the SacI site and the 3' end is beyond the end of the 4.5kb HindIII fragment. No homology has been found between predicted amino acid sequences and protein sequences present in the PIR and Swissprot databases.

Mendenhall, Alexander R. et al. (2011) International Worm Meeting "Factors Affecting the Mean, Variance and Predictive Power of a Lifespan-Predicting Biomarker."

Mendenhall, Alexander R., Lowe, Anita, Tedesco, Pat, Johnson, Thomas E., Taylor, Larry, Brent, Roger, Cypser, James R.

[International Worm Meeting, 2011]

Variation in gene expression among genetically identical individuals grown together in a uniform environment has been largely unexplored in C. elegans. In 2005, Rea et al. showed that the induced expression level of a transcriptional reporter of a small heatshock protein (Phsp-16.2::gfp) in young adult C. elegans could predict lifespan and thermotolerance. However, these studies used a single transgenic insertion (gpIs1), which contains approximately 450 copies of the transgene in a tandem array integrated into chromosome IV. To determine if different transgenic reporter alleles of the same gene show similar predictive capabilities and variance in expression, we generated several new transgenic lines expressing green and red fluorescent proteins under control of the hsp-16.2 promoter; these lines include three new single-copy reporters. Worm-to-worm variance in transgene expression is independent of locus, copy number, and fluor. Variance in GFP signal, measured either as worm-to-worm variance among individual worms or as cell-to-cell variance among matched intestine cells, is not significantly different between strains. The average expression profiles of the single-copy and tandem-array reporters display the most and least GFP in the exact same intestine cells; however, cell-to-cell measures are still preliminary and ongoing. Growth temperature affects the mean and variance of constitutively expressed reporters (Pvit-2::gfp and Pgpd-2::gfp). In contrast, mean and variance of the induced expression of Phsp-16.2::gfp are independent of growth temperature. Interestingly, we observed spontaneous, constitutive Phsp-16.2::gfp expression at 25deg C. Elimination or attenuation of specific signaling systems results in reduced worm-to-worm variance in Phsp-16.2::gfp expression. Thus, worm-to-worm variance in reporter expression is actively increased by intrinsic individual differences in and/or stochastic fluctuation of signal transduction between individuals. Finally, both single copy and tandem array transgenes predict thermotolerance and lifespan. Our studies demonstrate that the predictive capacity of the Phsp-16.2::gfp biomarker is not dependent on any specific transgene configuration. This abstract marks the beginning of a systems biology approach to dissect metazoan gene expression variance at the level of individual animals and cells to determine how single reporters can predict complex phenotype outcomes such as lifespan.

Maruyama IN et al. (1986) Worm Breeder's Gazette "a subtle neural connectivity mutant - unc-13."

Nawrocki LW, Maruyama IN

[Worm Breeder's Gazette, 1986]

The loci unc-13 and unc-15 are positioned very close together in the cluster of linkage group I. Both mutants have distinct phenotypes; unc- 15 (e73) worms are limp and paralyzed, while unc-13 (e450) worms are kinked and paralyzed. Mutations at the unc-15 locus affect the putative structural gene for paramyosin. Clones for unc-15 are available and have been used to map a contig that may be long enough to contain the unc-13 locus. A cosmid from the far end of this contig will be used to clone unc-13. Additional incentive to clone unc-13 has come from the isolation of a strain with a restriction site polymorphism from a TR679 background that affects both unc-13 and unc- 15 (see Roger Hoskins' report in this newsletter). In anticipation of cloning unc-13, we wished to identify the lesion causing its phenotype.

Jones D et al. (1992) Worm Breeder's Gazette "A Ubiquitin Fusion Gene from C. elegans"

Candido EPM, Jones D

[Worm Breeder's Gazette, 1992]

Eukaryotes in general have three types of ubiquitin genes: a polyubiquitin gene which encodes multiple tandem copies of ubiquitin transcribed as a polyprotein and two other genes which are fusions of ubiquitin with either a 52 aa or an 80aa ribosomal protein. The polyubiquitin gene from C. elegans was described a few years ago by Roger Graham (Mol. Cell. Biol. (1989)2: 268-271). Since then, we have been studying the regulation of this gene and searching for additional ubiquitin genes. Roger was able to isolate a clone for the 52 aa fusion using a protocol he described in WBG (1990) 11(2):69. A subclone of the ribosomal-protein encoding region was used to probe a YAC grid. A single positive was found, on chromosome III. The most notable aspect of the organization of this gene is the number and position of introns. Two introns occur in the ubiquitin portion of the gene. The position of the first is the same as that in four repeats of the polyubiquitin gene. (C. elegans is the only organism known to have introns in the polyubiquitin gene.) The second intron splits the two terminal glycine residues of ubiquitin. Neither of these intron positions coincides with those in any other 52 aa fusion gene [See Figure 1]. The sequence of a cDNA clone for this gene, isolated from Bob Barstead's lZAP library, shows that the message is trans-spliced. As an additional bonus, this gene has a recognizable TATA motif (unlike the poyubiquitin gene) which is 138 bp from the ATG start codon. We have produced and are currently injecting constructs of this gene. We expect it to be universally constitutive (since it encodes a ribosomal protein) and hope that the promoter may have some practical use. In addition, we may even find out why these ribosomal proteins are always made fused to ubiquitin and what the fate of the ubiquitin part of the fusion is. The search for the third ubiquitin gene continues

Schafer WR et al. (2000) European Worm Meeting "Regulation of calcium transients in the pharyngeal and vulval muscles"

Schafer WR, Kerr RA, Tsien RY

[European Worm Meeting, 2000]

Genetic and behavioral analysis has defined a number of signal transduction molecules that regulate the activity of the muscle cells involved in feeding and egg-laying behavior. To obtain understand more precisely the effects of these molecules on muscle cell physiology, we are using in vivo calcium imaging to visualize muscle calcium transients in live wild-type and mutant animals. We have expressed the FRET-based calcium-sensitive protein cameleon, developed in Roger Tsien's lab, in the pharyngeal and vulval muscles, and demonstrated that the calcium influx accompanying contraction can be reliably detected through ratiometric video-rate fluorescence imaging. Moreover, by analyzing the fluorescence traces of egl-19 calcium channel mutants previously shown by the Avery lab to affect the duration of the pharyngeal action potential, we have found that we can use cameleon to measure and distinguish effects of specific mutations on either the duration or the magnitude of the pharyngeal calcium influx. Using this approach, we have made the surprising discovery that unc-36 loss-of-function mutations, which eliminate the activity of the conserved large accessory (alpha-2) subunit of the calcium channel, cause a significant increase in the magnitude of the pharyngeal calcium transient. This suggests that the function of the alpha-2 subunit in muscle cells is to negatively regulate calcium influx through the channel. We have also expressed cameleon in the vulval muscles, and been similarly successful in detecting changes in fluorescence ratio associated with calcium transients. Preliminary results using this technique indicate that serotonin, a molecule known to function as a modulator of vulval muscle activity, dramatically increases the frequency of sub-threshold calcium transients (i.e. transients too small to induce an egg-laying event). Further analysis of vulval muscle calcium dynamics will be presented.