[
Worm Breeder's Gazette,
1993]
On two occasions we have run into problems trying to recombine linked markers with integrated high copy arrays of injected plasmids. jeIn2 is an integration of a
mec-3 -lacZfusionplasmid plus a 'Twitcher' marker plasmid into the right arm of chromosome IV. It has been possible to recombine this element with
mec-3 and
dpy-20 ,and these markers appear to be less than 5 map units to the right of the integrated array. However, we have not been able to generate true recombinants containing jeIn2 and the distal markers
ced-3 ,
ham-1 ,or
dpy-4 .From a jeIn2 + / +
dpy-4 heterozygote, occasional Dpy Twi animals can be isolated. Instead of finding that 1/3 of their Dpy Twi offspring are homozygous for the jeIn2 element, we find that all of the Twi animals have non-Twi progeny. To explain this, we invoke two ideas: 1) the integrated array suppresses recombination distal to itself, and 2) the integrated array can, at some frequency, excise and generate an unstable extrachromosomal array. The fate of the chromosome from which the array excises is unclear, as it is selected against when isolating these ''recombinants." We have observed apparent excision in crosses with jeIn2 and
ham-1 and with jeIn1 (
mec-7 -lacZon Chr. I) and
lin-17 .Both these arrays can undergo normal recombination with centrally located markers. Both arrays were generated by gamma ray-mediated integration of an extrachromosomal array. We have not examined oligonucleotide-generated arrays for recombination suppression. It is worth remembering that an integrated array that contains 100 copies of a 10kb plasmid would be 1 Mb in length, and it seems plausible that such an insertion could disrupt chromosome pairing. Also, since the arrays contain many repeated copies of one or two plasmids, they could undergo internal homologous recombination to generate an extrachromosomal circle. It might be possible to use large integrated arrays to look for sites that mediate chromosome pairing for recombination.
[
International Worm Meeting,
2017]
Extracellular vesicles are emerging as an important aspect of intercellular communication by delivering a parcel of proteins, lipids even nucleic acids to specific target cells over short or long distances (Maas 2017). A subset of C. elegans ciliated neurons release EVs to the environment and elicit changes in male behaviors in a cargo-dependent manner (Wang 2014, Silva 2017). Our studies raise many questions regarding these social communicating EV devices. Why is the cilium the donor site? What mechanisms control ciliary EV biogenesis? How are bioactive functions encoded within EVs? EV detection is a challenge and obstacle because of their small size (100nm). However, we possess the first and only system to visualize and monitor GFP-tagged EVs in living animals in real time. We are using several approaches to define the properties of an EV-releasing neuron (EVN) and to decipher the biology of ciliary-released EVs. To identify mechanisms regulating biogenesis, release, and function of ciliary EVs we took an unbiased transcriptome approach by isolating EVNs from adult worms and performing RNA-seq. We identified 335 significantly upregulated genes, of which 61 were validated by GFP reporters as expressed in EVNs (Wang 2015). By characterizing components of this EVN parts list, we discovered new components and pathways controlling EV biogenesis, EV shedding and retention in the cephalic lumen, and EV environmental release. We also identified cell-specific regulators of EVN ciliogenesis and are currently exploring mechanisms regulating EV cargo sorting. Our genetically tractable model can make inroads where other systems have not, and advance frontiers of EV knowledge where little is known. Maas, S. L. N., Breakefield, X. O., & Weaver, A. M. (2017). Trends in Cell Biology. Silva, M., Morsci, N., Nguyen, K. C. Q., Rizvi, A., Rongo, C., Hall, D. H., & Barr, M. M. (2017). Current Biology. Wang, J., Kaletsky, R., Silva, M., Williams, A., Haas, L. A., Androwski, R. J., Landis JN, Patrick C, Rashid A, Santiago-Martinez D, Gravato-Nobre M, Hodgkin J, Hall DH, Murphy CT, Barr, M. M. (2015).Current Biology. Wang, J., Silva, M., Haas, L. A., Morsci, N. S., Nguyen, K. C. Q., Hall, D. H., & Barr, M. M. (2014). Current Biology.