Forkhead transcription factors: Key players in development and metabolism

We have created a library of 2007 mutagenized Caenorhabditis elegans strains, each sequenced to a target depth of 15-fold coverage, to provide the research community with mutant alleles for each of the worm's more than 20,000 genes. The library contains over 800,000 unique single nucleotide variants (SNVs) with an average of eight nonsynonymous changes per gene and more than 16,000 insertion/deletion (indel) and copy number changes, providing an unprecedented genetic resource for this multicellular organism. To supplement this collection, we also sequenced 40 wild isolates, identifying more than 630,000 unique SNVs and 220,000 indels. Comparison of the two sets demonstrates that the mutant collection has a much richer array of both nonsense and missense mutations than the wild isolate set. We also find a wide range of rDNA and telomere repeat copy number in both sets. Scanning the mutant collection for molecular phenotypes reveals a nonsense suppressor as well as strains with higher levels of indels that harbor mutations in DNA repair genes and strains with abundant males associated with him mutations. All the strains are available through the Caenorhabditis Genetics Center and all the sequence changes have been deposited in WormBase and are available through an interactive website.

/pdf/the-million-mutation-project-a-new-approach-to-genetics-in-lxuh79zcnq.pdf

The Million Mutation Project: A new approach to genetics in Caenorhabditis elegans

The intestine is one of the major organs in C. elegans and is largely responsible for food digestion and assimilation as well as the synthesis and storage of macromolecules. In addition, the intestine is emerging as a powerful experimental system in which to study such universal biological phenomena as vesicular trafficking, biochemical clocks, stress responses and aging. The present chapter describes some of these many and varied properties of the C. elegans intestine: the embryonic cell lineage, intestine morphogenesis, structure and physiology of the intestinal cell and, finally, the transcription factor network controlling intestine development and function.

/pdf/the-c-elegans-intestine-31ioljciku.pdf

The C. elegans intestine.

Members of the transforming growth factor β (TGF-β) superfamily of cell signalling polypeptides have attracted much attention because of their ability, from nematodes to mammals, to control cellular functions that in turn, regulate embryo development and tissue homeostasis.

On the basis of structure similarities, the TGF-β members (ligands, receptors and Smads) are subdivided into TGF-β sensu stricto, bone morphogenetic proteins (BMP) and activins. Although BMP is the best characterized pathway in metazoans, recent findings in molluscs and non-bilateria as well as the analysis of nematode and arthropod genomes, demonstrate the early origin of these distinct subfamilies of ligands, receptors and Smads. This report analyses the large diversity of ligands, receptors and Smads in metazoans from cnidarians and molluscs to mammals.

The contribution of new data, mainly from the lophochotrozoan Crassostrea gigas and other organisms on the fringe of the ‘branded model organisms’, will help us to demonstrate that TGF-βs are probably the most ancestral active cytokines characterized at the molecular level in both Protostome and Deuterostome lineages.

Transforming growth factor-β-related proteins: an ancestral and widespread superfamily of cytokines in metazoans

Our understanding of the genetic mechanisms that underlie biological processes has relied extensively on loss-of-function (LOF) analyses LOF methods target DNA, RNA or protein to reduce or to ablate gene function By analysing the phenotypes that are caused by these perturbations the wild-type function of genes can be elucidated Although all LOF methods reduce gene activity, the choice of approach (for example, mutagenesis, CRISPR-based gene editing, RNA interference, morpholinos or pharmacological inhibition) can have a major effect on phenotypic outcomes Interpretation of the LOF phenotype must take into account the biological process that is targeted by each method The practicality and efficiency of LOF methods also vary considerably between model systems We describe parameters for choosing the optimal combination of method and system, and for interpreting phenotypes within the constraints of each method

/pdf/loss-of-function-genetic-tools-for-animal-models-cross-3s7e8caboo.pdf

Loss-of-function genetic tools for animal models: cross-species and cross-platform differences.

To study essential maternal gene requirements in the early C. elegans embryo, we have screened for temperature-sensitive, embryonic lethal mutations in an effort to bypass essential zygotic requirements for such genes during larval and adult germline development. With conditional alleles, multiple essential requirements can be examined by shifting at different times from the permissive temperature of 15°C to the restrictive temperature of 26°C. Here we describe 24 conditional mutations that affect 13 different loci and report the identity of the gene mutations responsible for the conditional lethality in 22 of the mutants. All but four are mis-sense mutations, with two mutations affecting splice sites, another creating an in-frame deletion, and one creating a premature stop codon. Almost all of the mis-sense mutations affect residues conserved in orthologs, and thus may be useful for engineering conditional mutations in other organisms. We find that 62% of the mutants display additional phenotypes when shifted to the restrictive temperature as L1 larvae, in addition to causing embryonic lethality after L4 upshifts. Remarkably, we also found that 13 out of the 24 mutations appear to be fast-acting, making them particularly useful for careful dissection of multiple essential requirements. Our findings highlight the value of C. elegans for identifying useful temperature-sensitive mutations in essential genes, and provide new insights into the requirements for some of the affected loci.

/pdf/a-survey-of-new-temperature-sensitive-embryonic-lethal-1s26rebfgp.pdf

A Survey of New Temperature-Sensitive, Embryonic-Lethal Mutations in C. elegans: 24 Alleles of Thirteen Genes

Genome Science: A Practical and Conceptual Introduction to Molecular Genetic Analysis in Eukaryotes

Biological invasions of exotic species pose a significant threat to local biodiversity and ecosystem services. Redistribution of plant species in particular disrupts key structure‐function relationships in the soil by altering the composition of microbial taxa. This study combines environmental DNA analysis with a goat‐grazing intervention to investigate the taxonomic structure of soil microbiomes interacting with the invasive plant, Rhamnus cathartica (common Buckthorn). 16S (515F/806R) metabarcodes were amplified from soil DNA harvested before and after a goat‐grazing regimen on buckthorn‐invaded and control plots. Samples were sequenced on an Illumina iSeq, then analyzed using the DNA Subway Purple Line (QIIME 2.0) to process sequencing reads and calculate diversity indices. PCoA visualizations of Jaccard Beta diversity patterns revealed differences in community composition between buckthorn and control soils, but no effects from goat grazing. Five dominant taxa were identified in no‐buckthorn control soils, accounting for 35 +/‐ 9.6 SD% of the average relative abundance of total sequences. Genus DA101 notably demonstrated the largest decrease in average relative abundance in the buckthorn and goat‐grazed soils (% decrease = 6.4 +/‐ 2 SD), whereas the evenness of other taxa increased. Overall, buckthorn soil showed significant increases in Pielou’s taxonomical evenness (Kruskal‐Wallis H(2, N=29) = 10.9; p=0.004), but goat grazing had no impact on this score (p>0.01). Faith’s phylogenetic diversity scores showed no significant differences between buckthorn, goat‐grazed, and control soils (Kruskal‐Wallis H(2, N=31) = 2.2; p=0.32). Taken together, these preliminary results illustrate that buckthorn increases taxonomical evenness but has no effect on overall phylogenetic diversity of soil communities. Moreover, buckthorn goat‐grazing had little impact on soil community structure. Future studies will investigate the role of DA101 on buckthorn suppression as a possible mitigation strategy for this invasive species.

eDNA Analysis of Goat‐Grazed Rhamnus cathartica Soil Microbial Communities

Analyzing the impacts of tobacco mosaic virus on the microbial diversity of Nicotianabenthamiana

Wildfires have continued to increase in frequency and severity in Southern California due in part to climate change. To gain a further understanding of microbial soil communities’ response to fire and functions that may enhance post-wildfire resilience, soil fungal and bacterial microbiomes were studied from different wildfire areas in the Gold Creek Preserve within the Angeles National Forest using 16S, FITS, 18S, 12S, PITS, and COI amplicon sequencing. Sequencing datasets from December 2020 and June 2021 samplings were analyzed using QIIME2, ranacapa, stats, vcd, EZBioCloud, and mixomics. Significant differences were found among bacterial and fungal taxa associated with different fire areas in the Gold Creek Preserve. There was evidence of seasonal shifts in the alpha diversity of the bacterial communities. In the sparse partial least squares analysis, there were strong associations (r > 0.8) between longitude, elevation, and a defined cluster of Amplicon Sequence Variants (ASVs). The Chi-square test revealed differences in fungi–bacteria (F:B) proportions between different trails (p = 2 × 10−16). sPLS results focused on a cluster of Green Trail samples with high elevation and longitude. Analysis revealed the cluster included the post-fire pioneer fungi Pyronema and Tremella. Chlorellales algae and possibly pathogenic Fusarium sequences were elevated. Bacterivorous Corallococcus, which secretes antimicrobials, and bacterivorous flagellate Spumella were associated with the cluster. There was functional redundancy in clusters that were differently composed but shared similar ecological functions. These results implied a set of traits for post-fire resiliency. These included photo-autotrophy, mineralization of pyrolyzed organic matter and aromatic/oily compounds, potential pathogenicity and parasitism, antimicrobials, and N-metabolism.

https://www.mdpi.com/2076-2607/10/6/1218/pdf?version=1655199670

The Functional Biogeography of eDNA Metacommunities in the Post-Fire Landscape of the Angeles National Forest

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