Stein, Samantha A. et al. (2022) MicroPubl Biol

Maine, Eleanor M., Smith, Harold E., Lissemore, James L., O'Connell, Kevin F., Stein, Samantha A., Zucaro, Olivia F., Spoerke, Jill M.

[MicroPubl Biol, 2022]

Germline stem cell proliferation in C. elegans requires activation of the GLP-1/Notch receptor, which is located on the germline plasma membrane and encoded by the glp-1 gene. We previously identified several genes whose products directly or indirectly promote activity of the GLP-1 signaling pathway by finding mutations that enhance the germline phenotype of a glp-1(ts) allele, glp-1(bn18) . Here, we report phenotypic and molecular analysis of a new ekl-1 allele, ekl-1(om92) , that enhances the glp-1(bn18) phenotype. ekl-1(om92) is a 244 bp deletion predicted to generate a frameshift and premature termination codon, yielding a severely truncated protein, suggesting it is a null allele.

Stein KK et al. (2015) WormBook "The C. elegans eggshell."

Stein KK, Golden A

[WormBook, 2015]

In all animals, oocytes are surrounded by an extracellular matrix upon fertilization. This matrix serves similar purposes in each animal. It functions to mediate sperm binding, to prevent polyspermy, to control the chemical environment of the embryo, and to provide physical protection to the embryo as it developes. The synthesis of the C. elegans matrix, or eggshell, begins when the oocyte enters the spermatheca and is fertilized by a single sperm. The process of eggshell synthesis is thought to take place during the completion of the maternal meiotic divisions such that the multi-layered eggshell is completed by anaphase II. The synthesis of the eggshell occurs in a hierarchical pattern in which the outermost layers are synthesized first in order to capture and retain the innermost layers as they form. Recent studies have revealed that the lipid-rich permeability barrier is distinct from the outer trilaminar eggshell. These new findings alter our previous understanding of the eggshell. This chapter aims to define each of the eggshell layers and the molecules that are known to play significant roles in their formation.

Stein JA et al. (1984) Worm Breeder's Gazette "a computer program for organizing a clone collection."

Emmons SW, Stein JA

[Worm Breeder's Gazette, 1984]

We have written a computer program for storage and retrieval of information about the recombinant DNA collection held by a laboratory. For each clone, information such as clone name, cloning vector, cloner, date cloned, notebook page, storage location, etc., is entered. The cloned insert is identified by a serially-assigned fragment number and by a restriction map. Wherever a given fragment occurs in the collection it is identified by its fragment number, and all restriction mapping data for that fragment is held together in a single storage location and kept updated. Data regarding interrelationships among fragments in the collection, such as which are subclones of others, are also held. The stored information can be searched and listed in a variety of ways. The program is modular in design and can be readily modified and expanded. It is written in Basic for a Dec RT-11 operating system ( PDP-11 computer or equivalent) and should be easily adaptable to other systems. We encourage other laboratories to use it. The program anticipates multiple laboratory use by prefixing each fragment number with a laboratory number. By this means each cloned segment of the genome is given a unique name. Eventually there will undoubtedly be a need for a centralized listing of information about cloned fragments of the C. elegans genome. We suggest that a centralized listing could be a subset of the information stored in laboratory based listings such as this one. For a free interchange of information among laboratories and between laboratories and a centralized data base to be possible, a uniform system of nomenclature and computerized format will have to be adopted. We have written this program in order to gain some experience with the use of a laboratory based data bank that hopefully will be helpful in designing a widely used system. Please contact us if you are interested in using this program. We would be glad to help you adapt it to your computer system.

Stein LD (1999) Trends Genet "Internet access to the C. elegans genome."

Stein LD

[Trends Genet, 1999]

In December 1998, the genome of the small soil-dwelling nematode Caenorhabditis elegans became the most recent model to fall to the collective efforts of the Genome Project, its complete 97 Mbp genome taking its place alongside those of Saccharomyces cerevisiae, Escherichia coli and numerous other microorganisms. The availability of the C. elegans sequence means that, for the first time, the complete genome of a fully functional multicellular animal is available to the scientific community, along with a rich infrastructure of genetic, behavioral, physiological and developmental data about the organism.

Stein KC et al. (2022) Nature "Ageing exacerbates ribosome pausing to disrupt cotranslational proteostasis."

Frydman J, Rainbolt TK, van der Lienden J, Morales-Polanco F, Stein KC

[Nature, 2022]

Ageing is accompanied by a decline in cellular proteostasis, which underlies many age-related protein misfolding diseases^1,2. Yet, how ageing impairs proteostasis remains unclear. As nascent polypeptides represent a substantial burden on the proteostasis network³, we hypothesized that altered translational efficiency during ageing could help to drive the collapse of proteostasis. Here we show that ageing alters the kinetics of translation elongation in both Caenorhabditis elegans and Saccharomyces cerevisiae. Ribosome pausing was exacerbated at specific positions in aged yeast and worms, including polybasic stretches, leading to increased ribosome collisions known to trigger ribosome-associated quality control (RQC)^4-6. Notably, aged yeast cells exhibited impaired clearance and increased aggregation of RQC substrates, indicating that ageing overwhelms this pathway. Indeed, long-lived yeast mutants reduced age-dependent ribosome pausing, and extended lifespan correlated with greater flux through the RQC pathway. Further linking altered translation to proteostasis collapse, we found that nascent polypeptides exhibiting age-dependent ribosome pausing in C. elegans were strongly enriched among age-dependent protein aggregates. Notably, ageing increased the pausing and aggregation of many components of proteostasis, which could initiate a cycle of proteostasis collapse. We propose that increased ribosome pausing, leading to RQC overload and nascent polypeptide aggregation, critically contributes to proteostasis impairment and systemic decline during ageing.

LD Stein et al. (1999) International C. elegans Meeting "AcePerl: A New Face for Ace"

LD Stein, J Thierry-Mieg, R Durbin

[International C. elegans Meeting, 1999]

Stein, Paul Neal et al. (2013) International Worm Meeting "ROS and antioxidant interaction in C. elegans."

Stein, Paul Neal, LaMunyon, Craig W.

[International Worm Meeting, 2013]

Reactive oxygen species (ROS) are thought to be partially responsible for aging and senescence, but the mechanisms by which ROS participate in aging are not entirely clear. Antioxidants may neutralize harmful ROS, and they have been promoted as a means of therapy. In order to explore the relationship between ROS and antioxidants, we assessed protein damage, antioxidant linked ROS suppression and native antioxidant expression under various treatment conditions. We had earlier shown that worms harboring the uaDf5 mitochondrial DNA deletion produced elevated hydrogen peroxide and experienced shorter lifespans. The lifespan deficit was reversed by treatment with the antioxidant EUK-134 but not by coenzyme Q10. Here, we tested both antioxidants on uaDf5 worms and worms treated with the ROS-inducing mitochondrial toxin paraquat. We detected no effect of the treatment on protein damage measured as the quenching of fluorescence of a Pmyo-3::GFP transcriptional fusion. We did find changes in hydrogen peroxide production, assayed using the fluorescent dye DCF. The expression of a Psod-3::GFP transcriptional fusion did not change with treatment, although changes in transcript abundance of a host of sod genes were detected by RT-PCR. Our combined results show that sod gene transcription responds to variation in ROS and treatment with EUK-134.

Stein GM et al. (2012) Front Genet "The Intersection of Aging, Longevity Pathways, and Learning and Memory in C. elegans."

Stein GM, Murphy CT

[Front Genet, 2012]

Our understanding of the molecular and genetic regulation of aging and longevity has been greatly augmented through studies using the small model system, C. elegans. It is important to test whether mutations that result in a longer life span also extend the health span of the organism, rather than simply prolonging an aged state. C. elegans can learn and remember both associated and non-associated stimuli, and many of these learning and memory paradigms are subject to regulation by longevity pathways. One of the more distressing results of aging is cognitive decline, and while no gross physical defects in C. elegans sensory neurons have been identified, the organism does lose the ability to perform both simple and complex learned behaviors with age. Here we review what is known about the effects of longevity pathways and the decline of these complex learned behaviors with age, and we highlight outstanding questions in the field.

Kathryn K Stein et al. (2005) International Worm Meeting "Paternal effect lethal mutations in C. elegans"

Kathryn K Stein, Andy Golden

[International Worm Meeting, 2005]

In preparation for fertilization and embryogenesis a variety of maternal factors are produced and stored in the oocyte. These factors supply the early embryo with the components needed for several cell divisions until zygotic transcription commences. In contrast to the large, well-furnished oocyte, the primary contribution of the sperm is its nuclear material, which restores a diploid state to the embryo. There is evidence that sperm also contain specific paternal factors, which contribute to embryogenesis. Absence of these sperm factors results in paternal effect embryonic lethality. Lethality may be due to defects in egg activation, paternal chromosome decondensation, or chromosome function. To date, one strict paternal effect lethal (pel) mutant is known in C. elegans, the product of the gene spe-11. In the absence of functional SPE-11, embryogenesis fails at early stages. SPE-11 is a novel cytoplasmic protein that is supplied to the embryo exclusively by the sperm. SPE-11 is localized to the nucleus, although its putative role there is not known. Ectopic expression of SPE-11 by the oocyte in spe-11 hermaphrodites is sufficient to rescue the embryonic defect (Browning and Strome, 1996; Hill et al., 1989). In this study our aim is to further understand the paternal role of SPE-11. We will use several approaches to this end, one of which is to identify suppressors of spe-11 in a genetic screen. In spe-11 animals very few live progeny are produced. In the presence of a mutation that suppresses spe-11, embryonic lethality will occur at a reduced level and the number of live progeny will increase. Using this criterion, we have identified several potential suppressors. These suppressors are expected to encode proteins that function in the same pathway as SPE-11, which will contribute to our understanding of the role of the sperm in embryogenesis. We are also interested in identifying and characterizing other putative paternal effect candidates. In C. elegans, previous genetic screens have identified mutants in which embryonic lethality can be rescued by wild type males, indicative of a sperm defect. We are currently assessing if these mutants are true paternal effect lethals. We will confirm that mutant sperm produce dead embryos, even when fertilizing wild type oocytes. Our next goal will be to determine the molecular identity of these proteins.

Stein P et al. (2003) RNA "RNAi: mammalian oocytes do it without RNA-dependent RNA polymerase."

Svoboda P, Anger M, Stein P, Schultz RM

[RNA, 2003]

Studies in mutant organisms deficient in RNA interference (RNAi) and related post-transcriptional gene silencing implicated a role for a single class of RNA-dependent RNA polymerases (RdRp). Nevertheless, sequence homologs to these RdRps have not been found in coelomate organisms such as Drosophila or mammals. This lack of homologous sequences does not exclude that an RdRp functions in RNAi in these organisms because an RdRp could be acquired by horizontal transfer from an RNA virus. In fact, such a sequence is found in mice (Aquarius) and we observe that it is expressed in mouse oocytes and early embryos, which exhibit RNAi. We report here that cordycepin, an inhibitor of RNA synthesis, does not prevent Mos double-strand RNA (dsRNA) to target endogenous Mos mRNA in mouse oocytes and that targeting a chimeric Mos-EGFP mRNA with dsRNA to EGFP does not reduce the endogenous Mos mRNA, but does target the chimeric mRNA. These results indicate that an RdRp is not involved in dsRNA-mediated mRNA degradation in mammalian oocytes, and possibly in mammals in general, and therefore that only homologous sequences to the dsRNA are targeted for degradation.