Albritton, Sarah et al. (2017) International Worm Meeting "Long-distance cooperation and hierarchy between recruitment sites specify the X chromosome for dosage compensation."

Kranz, Anna-Lena, Winterkorn, Lara, Ercan, Sevinc, Albritton, Sarah, Street, Lena

[International Worm Meeting, 2017]

Dosage compensation mechanisms specifically target gene regulatory complexes to the X chromosomes for transcriptional regulation. However, it remains unclear how X is specified, as the DNA sequence motifs shown to be important for binding of dosage compensation machineries are themselves not X-specific. Here, we analyzed binding of the C. elegans dosage compensation complex (DCC) through a series of experiments that include deletion and ectopic insertion of recruitment site sequences. Our data suggest that DCC recruitment is initiated by a small number of primary recruitment sites characterized by clusters of the 12-bp recruitment motif and overlap with high occupancy transcription factor target (HOT) regions, two features that fully explain X-specificity. SDC-2, the protein essential for hermaphrodite-specific recruitment of the DCC during early embryogenesis, is required to maintain open chromatin specifically at the primary recruitment sites whose DNA sequence encodes for high intrinsic nucleosome occupancy. Along the X, the primary recruitment sites are interspersed by secondary, weaker DCC recruitment sites. While insertion of a secondary recruitment element on an autosome failed to fully recruit the DCC, the same element was capable of recruitment at an ectopic locus on the X, suggesting that the function of the weaker recruitment sites are X-dependent. On the autosome, insertion of multiple recruitment elements in tandem or at a distance (>30 kb) increased DCC recruitment, demonstrating that recruitment sites cooperate over long distances. On the X, deletion of single recruitment sites resulted in reduced DCC binding across several megabases flanked by topologically associating domain (TAD) boundaries, suggesting that DCC recruitment and spreading occurs within defined X chromosomal domains. Our work illustrates a fundamental strategy for specifically targeting large chromosomal domains for co-regulation, which involves hierarchy and long-distance cooperativity between functional genomic elements.

Qian W et al. (2008) Genome Res "Evolutionary dynamics of nematode operons: easy come, slow go."

Zhang J, Qian W

[Genome Res, 2008]

Operons are widespread in prokaryotes, but are uncommon in eukaryotes, except nematode worms, where approximately 15% of genes reside in over 1100 operons in the model organism Caenorhabditis elegans. It is unclear how operons have become abundant in nematode genomes. The "one-way street" hypothesis asserts that once formed by chance, operons are very difficult to break, because the breakage would leave downstream genes in an operon without a promoter, and hence, unexpressed. To test this hypothesis, we analyzed the presence and absence of C. elegans operons in Caenorhabditis briggsae, Caenorhabditis remanei, and Caenorhabditis brenneri, using Pristionchus pacificus and Brugia malayi as outgroups, and identified numerous operon gains and losses. Coupled with experimental examination of trans-splicing patterns, our comparative genomic analysis revealed diverse molecular mechanisms of operon losses, including inversion, insertion, and relocation, but the presence of internal promoters was not found to facilitate operon losses. In several cases, the data allowed inference of mechanisms by which downstream genes are expressed after operon breakage. We found that the rate of operon gain is approximately 3.3 times that of operon loss. Thus, the evolutionary dynamics of nematode operons is better described as "easy come, slow go," rather than a "one-way street." Based on a mathematic model of operon gains and losses and additional assumptions, we projected that the number of operons in C. elegans will continue to rise by 6%-18% in future evolution before reaching equilibrium between operon gains and losses.

Jimenez, David et al. (2021) International Worm Meeting "Condensin DC spreads linearly and bidirectionally from recruitment sites to create loop-anchored TADs in C. elegans"

Street, Lena, Kim, Jun, Kramer, Maxwell, Zhang, Bo, Winterkorn, Lara, Ragipani, Bhavana, Ercan, Sevinc, Jimenez, David, Albritton, Sarah

[International Worm Meeting, 2021]

Condensins are molecular motors that compact DNA for chromosome segregation and gene regulation. In vitro experiments have begun to elucidate the mechanics of condensin function but how condensin loading and translocation along DNA controls eukaryotic chromosome structure in vivo remains poorly understood. To address this question, we took advantage of a specialized condensin, which organizes the 3D conformation of X chromosomes to mediate dosage compensation (DC) in C. elegans. Condensin DC is recruited and spreads from a small number of recruitment elements on the X chromosome (rex). We found that ectopic insertion of rex sites on an autosome leads to bidirectional spreading of the complex over hundreds of kilobases. On the X chromosome, strong rex sites contain multiple copies of a 12-bp sequence motif and act as TAD borders. Inserting a strong rex site and ectopically recruiting the complex on the X chromosome or an autosome creates a loop-anchored TAD. However, unlike the CTCF system, which controls TAD formation by cohesin, direction of the 12-bp motif does not control the specificity of loops. In an X;V fusion chromosome, condensin DC linearly spreads and increases 3D DNA contacts, but fails to form TADs in the absence of rex sites. Finally, we provide in vivo evidence for the loop extrusion hypothesis by targeting multiple dCas9-Suntag complexes to an X chromosome repeat region. Consistent with linear translocation along DNA, condensin DC accumulates at the block site. Together, our results support a model whereby strong rex sites act as insulation elements through recruitment and bidirectional spreading of condensin DC molecules and form loop-anchored TADs.

Mello CC et al. (1990) Worm Breeder's Gazette "Worm Injection Setup and Parts List"

Mello CC, Ambros VR

[Worm Breeder's Gazette, 1990]

The accompanying diagram shows the system which we use to pressurize our microinjection setup. The key feature of this system is the three way ball valve through which pressure at the needle is applied and then rapidly relieved during each injection. Pressure must be off between injections because the high flow associated with optimal injections will float animals off the pad before the needle can be inserted. It may be possible to adapt your current system to include such a valve, or to construct a similar system with other parts. [See Figure 1] 1) Leitz microinstrument collar (part no. 520145). Holds the injection needle. 2) 3/32' outside diameter (1/32' I.D.) Tygon tubing. This tubing, once stretched, will fit snugly down the opening in the back of the microinstrument collar. This junction is taped with electrical tape. The tape keeps this joint from getting worked loose during the frequent handling of the microtool collar. The tape should not be necessary to make the pressure seal. 3) 21g metal tube (cut off of a syringe needle). Fitted halfway within the 3/32' tubing, the protruding portion is inserted into the 1/16' tubing and serves as a connecter. This connection is also wrapped with electrical tape. 4) 1/16' polyethylene tubing (rigid plastic or nylon tubing works best with the Swagelok adapters). Note; if you plan to HF treat your needles (Worm Breeders Gazette 11-1:18), then this tubing should be long enough to reach a nearby dissecting scope. S) Swagelok reducer (part no. B-100-R-2). Connects the 1/16' tubing to the Swagelok 1/8 connector which comes with the three way valve. 6) Swagelok three way ball valve (part no. B-41XS2). Should be mounted or clamped in place. This valve is used during each injection to apply pressure at the needle. It should be placed within easy reach but should not be on the vibration free table. 7) Swagelok male connector (part no. B-100-1-4). This connector threads into the 1/4 NPT female socket of the STREET TEE. 8) CAJON STREET TEE connector (part no. B-4-ST). Male end threads into the pressure regulator. Female ends accept the tubing connector and the pressure release valve. 9) Swagelok toggle operated shut off valve (B-1GM4-S4). This valve opens the system to allow a quick release of pressure. This is necessary when using the pressure regulator to make downward adjustments in pressure, for example if there is too much flow at the needle. 10) Pressure regulator. Must have a 1/4 NPT Female thread in order to accept the STREET TEE described above. Allows the pressure to be increased or decreased. Pressures required for adequate flow range from 10- to 100-lbs/sq.in. depending on the needle; higher pressures tend to blow connections or to propel the needle out of the holder at high velocity. 11) Nitrogen tank. Should be positioned so that the regulator handle can be reached easily without getting up during injection. Happy injecting!!

Grishok A et al. (2000) Science "Genetic requirements for inheritance of RNAi in C. elegans."

Grishok A, Tabara H, Mello CC

[Science, 2000]

In Caenorhabditis elegans, the introduction of double-stranded RNA triggers sequence-specific genetic interference (RNAi) that is transmitted to offspring. The inheritance properties associated with this phenomenon were examined. Transmission of the interference effect occurred through a dominant extragenic agent. The wild-type activities of the RNAi pathway genes rde-1 and rde-4 were required for the formation of this interfering agent but were not needed for interference thereafter. In contrast, the rde-2 and mut-7 genes were required downstream for interference. These findings provide evidence for germ line transmission of an extragenic sequence-specific silencing factor and implicate rde-1 and rde-4 in the formation of the inherited agent.AD - Program in Molecular Medicine, Department of Cell Biology, University of Massachusetts Cancer Center, Two Biotech Suite 213, 373 Plantation Street, Worcester, MA 01605, USA.FAU - Grishok, AAU - Grishok AFAU - Tabara, HAU - Tabara HFAU - Mello, C CAU - Mello CCLA - engID - GM58800/GM/NIGMSPT - Journal ArticleCY - UNITED STATESTA - ScienceJID - 0404511RN - 0 (DNA Transposable Elements)RN - 0 (Helminth Proteins)RN - 0 (RNA, Double-Stranded)RN - 0 (RNA, Helminth)RN - 0 (rde1 protein)SB - IM

Trifuoggi M et al. (2018) Environ Monit Assess "Topsoil and urban dust pollution and toxicity in Taranto (southern Italy) industrial area and in a residential district."

Buric P, Palumbo A, Tommasi F, Guida M, Trifuoggi M, Thomas PJ, Crisci A, D'Ambra L, Toscanesi M, Oral R, Siciliano A, Gravina M, Pagano G, Mozzillo M, Lyons DM

[Environ Monit Assess, 2018]

Adverse environmental conditions in the Taranto area (southern Italy) were investigated in studies of air, marine sediment, and human health. The present study aimed at providing unprecedented information on soil pollution and toxicity in a set of sites around recognized pollution sources in the Taranto area, since previous studies were focused on marine or air pollution, or on human health effects. The investigated area included a steel foundry and a power plant, as well as some sites located in an adjacent neighborhood. Surface soil samples and urban dust were collected and submitted to inorganic and organic analyses and tested for toxicity in two invertebrate bioassay models; a sea urchin (Sphaerechinus granularis) and an annelid (Caenorhabditis elegans). Inorganic analysis was carried out using ICP-MS for elemental composition for a total of 34 elements, whose levels were evaluated as a function of bioassay data analyzed through principal component analysis (PCA). Other analyses included asbestos search by powder X-ray diffraction (PXRD) and organic analysis for polycyclic aromatic hydrocarbons (PAHs) and aliphatic compounds (C10-C40). Toxicity bioassays were carried out on a sea urchin (Sphaerechinus granularis), and an annelid (Caenorhabditis elegans). Sea urchin bioassays evaluated effects of topsoil or street dust sample exposures (0.1 to 0.5% dry wt/vol) on developing embryos and on sperm, and scored as (a) % developmental defects, (b) inhibition of fertilization success and offspring damage, and (c) frequencies of mitotic aberrations. C. elegans mortality assay displayed significant toxicity associated with soil samples. The overall effects of samples showed very high toxicity at four out of nine sites. These effects were consistent with the highest levels measured for metals and PAHs. Further studies of health effects related to dust exposures in residential areas are warranted. Graphical abstract .

Squire MD et al. (1994) Worm Breeder's Gazette "Cloning the Inositol 1,4,5 Triphosphate Receptor."

Baylis HA, Squire MD, Sattelle DB

[Worm Breeder's Gazette, 1994]

CLONING THE INOSITOL 1,4,5 TRISPHOSPHATE RECEPTOR. Howard Baylis, Mike Squire and David Sattelle The Babraham Institute Laboratory of Molecular Signalling, Department of Zooloqy, University of Cambridge, Downing Street, Cambridge UK. The intracellular second messenger inositol 1,4,5 trisphosphate (InsP3) binds to a specific receptor (InsP3R) that releases Ca2+ from intracellular storage sites, thereby modulating various Ca2+-associated proteins in cells (Mikoshiba et al 1993 Ann. NY Acad Sci 707, 178-197). As part of a project to study inositol 1,4,5 trisphosphate (InsP3) signalling using C. elegans we have been attempting to clone the nematode InsP3 receptor. Using degenerate primers to two sequences conserved amongst InsP3 receptors in mammals, Xenopus and Drosophila we were able to amplify a 240bp (IP240) fragment of the coding sequence, either from genomic DNA or from random primed 1st strand cDNA. In addition, a search of the NCBI EST database with the Drosophila sequence revealed that one of Yuji Kohara's (WBG 13 20-21) cDNAs (yk33g8) encoded 445 bp covering the end of the gene and part or all of the 3' untranslated region. This clone was clearly different from cDNA clones encoding the known ryanodine receptor (eg yk4h3). The amino acid sequences encoded by the two sequences are clearly much closer to InsP3 receptors than to the ryanodine receptors. Thus the evidence to date suggests that this is the InsP3 receptor. An alignment of one part of the C. elegans sequence and InsP3 receptor sequences from other organisms (figure 1) shows that some of the residues which are identical in all the other sequences are not conserved in C. elegans. Thus the sequence of this molecule may provide insights into structure and function of the protein. We have recently obtained a PCR product covering the 3kb between the two fragments and are currently working towards obtaining a full length cDNA (expected to be in the region of 8-9kb) and a genomic clone. From here we plan to study the function of the receptor using C. elegans genetics and in vitro expression and electrophysiology. We are indebted to Drs Katsuhiko Miknshiba Teiichi Furuichin, Yuji Kohara and John Fleming for their assistance during this study.