Protocol for the Electroporation of Neurospora Spheroplasts

A protocol for electroporating Neurospora strains is described. This protocol uses spheroplasts for transformation rather than germinated conidia with enzyme-weakened walls. High efficiency transformation is obtained with this system even with batches of Novozyme234 which worked poorly in the PEG based protocol

The Neurospora crassa dfg5 and dcw1 Genes Encode α-1,6-Mannanases That Function in the Incorporation of Glycoproteins into the Cell Wall

The covalent cross-linking of cell wall proteins into the cell wall glucan/chitin matrix is an important step in the biogenesis of the fungal cell wall. We demonstrate that the Neurospora crassa DFG5 (NCU03770) and DCW1 (NCU08127) enzymes function in vivo to cross-link glycoproteins into the cell wall. Mutants lacking DFG5 or DCW1 release slightly elevated levels of cell wall proteins into their growth medium. Mutants lacking both DFG5 and DCW1 have substantially reduced levels of cell wall proteins in their cell walls and release large amounts of known cell wall proteins into the medium. DFG5 and DCW1 are members of the GH76 family of glycosyl hydrolases, which have specificity to recognize and cleave α-1,6-mannans. A model for incorporation of glycoproteins into the cell wall through the α-1,6-mannan core of the N-linked galactomannan is presented. In this model, DFG5 and DCW1 recognize the N-linked galactomannan present on glycoproteins and cross-link it into the cell wall glucan/chitin matrix

Use of the Neurospora tyrosinase gene as a reporter gene in transformation experiments

Reporter gene systems have been developed to allow investigators to visually identify transformed cells and to follow the transcriptional activity from promoter regions preceding the reporter genes. These reporter genes encode protein products which can be easily assayed and which can be strained for in the transformed cell

Production of tyrosinase defective mutants of Neurospora crassa

We have produced mutants defective in tyrosinase activity using the RIP procedure (Selker et al. 1987 Cell 51:741-752, Marathe et al. 1990 Mol. Cell. Biol. 10:2638-2644). The tyrosinase gene has been shown to be a useful reporter in gene expression experiments (Kothe et al. 1993 FGN 40: 43-45). The availability of tyrosinase mutants will assist in these studies and will also be of use for future protein engineering experiments on tyrosinase (Fuentes et al. 1993 FGN 40:38-39

The ham-5, rcm-1 and rco-1 genes regulate hyphal fusion in Neurospora crassa

Mutants of Neurospora crassa unable to participate in vegetative hyphal fusion (anastomosis) were isolated and characterized. From this analysis, three genes, rcm-1, rco-1 and ham-5, were identified and shown to be required for hyphal fusion. The rcm-1 and rco-1 genes are homologues of the Saccharomyces cerevisiae SSN6 and TUP1 genes, which encode a dimeric transcription factor in yeast. We demonstrate that in N. crassa the rcm-1 and rco-1 genes are required for hyphal fusion and normal hyphal morphology, and influence both asexual and sexual development. The ham-5 gene encodes a 1686 amino acid protein with two putative WD40 domains, which might participate in protein–protein interactions. ham-5 deletion mutants had a reduced rate of hyphal extension and altered hyphal morphology, and were unable to produce the conidial anastomosis tubes that are required for hyphal fusion during colony initiation

NAFTA Chapter 11 as Supraconstitution

More and more legal scholars are turning to constitutional law to make sense of the growth of transnational and international legal orders. They often employ constitutional terminology loosely, in a bewildering variety of ways, with little effort to clarify their analytical frameworks or acknowledge the normative presuppositions embedded in their analysis. The potential of constitutional analysis as an instrument of critique of transnational legal orders is frequently lost in methodological confusion and normative controversy. An effort at clarification is necessary. We propose a functional approach to supraconstitutional analysis that applies across issue areas, accommodates variation in kinds and degrees of supraconstitutionalization, recognizes its simultaneously domestic and transnational character, and reflects its uneven incidence and impacts. We apply this framework to NAFTA to consider whether and how it superimposes a supraconstitutional legal order on member states\u27 domestic constitutional orders. We show that the main thrust of this contemporary supraconstitutional project is to restructure state and international political forms to promote market efficiency and discipline, enable free capital movement, confer privileged rights of citizenship and representation on corporate capital, insulate key aspects of the economy from state interference, and constrain democratic decision-making

Ecological Adaptation of Diverse Honey Bee (Apis mellifera) Populations

BACKGROUND: Honey bees are complex eusocial insects that provide a critical contribution to human agricultural food production. Their natural migration has selected for traits that increase fitness within geographical areas, but in parallel their domestication has selected for traits that enhance productivity and survival under local conditions. Elucidating the biochemical mechanisms of these local adaptive processes is a key goal of evolutionary biology. Proteomics provides tools unique among the major 'omics disciplines for identifying the mechanisms employed by an organism in adapting to environmental challenges. RESULTS: Through proteome profiling of adult honey bee midgut from geographically dispersed, domesticated populations combined with multiple parallel statistical treatments, the data presented here suggest some of the major cellular processes involved in adapting to different climates. These findings provide insight into the molecular underpinnings that may confer an advantage to honey bee populations. Significantly, the major energy-producing pathways of the mitochondria, the organelle most closely involved in heat production, were consistently higher in bees that had adapted to colder climates. In opposition, up-regulation of protein metabolism capacity, from biosynthesis to degradation, had been selected for in bees from warmer climates. CONCLUSIONS: Overall, our results present a proteomic interpretation of expression polymorphisms between honey bee ecotypes and provide insight into molecular aspects of local adaptation or selection with consequences for honey bee management and breeding. The implications of our findings extend beyond apiculture as they underscore the need to consider the interdependence of animal populations and their agro-ecological context

SARS-CoV-2 susceptibility and COVID-19 disease severity are associated with genetic variants affecting gene expression in a variety of tissues

Variability in SARS-CoV-2 susceptibility and COVID-19 disease severity between individuals is partly due to genetic factors. Here, we identify 4 genomic loci with suggestive associations for SARS-CoV-2 susceptibility and 19 for COVID-19 disease severity. Four of these 23 loci likely have an ethnicity-specific component. Genome-wide association study (GWAS) signals in 11 loci colocalize with expression quantitative trait loci (eQTLs) associated with the expression of 20 genes in 62 tissues/cell types (range: 1:43 tissues/gene), including lung, brain, heart, muscle, and skin as well as the digestive system and immune system. We perform genetic fine mapping to compute 99% credible SNP sets, which identify 10 GWAS loci that have eight or fewer SNPs in the credible set, including three loci with one single likely causal SNP. Our study suggests that the diverse symptoms and disease severity of COVID-19 observed between individuals is associated with variants across the genome, affecting gene expression levels in a wide variety of tissue types

Capacity shortfalls hinder the performance of marine protected areas globally

Marine protected areas (MPAs) are increasingly being used globally to conserve marine resources. However, whether many MPAs are being effectively and equitably managed, and how MPA management influences substantive outcomes remain unknown. We developed a global database of management and fish population data (433 and 218 MPAs, respectively) to assess: MPA management processes; the effects of MPAs on fish populations; and relationships between management processes and ecological effects. Here we report that many MPAs failed to meet thresholds for effective and equitable management processes, with widespread shortfalls in staff and financial resources. Although 71% of MPAs positively influenced fish populations, these conservation impacts were highly variable. Staff and budget capacity were the strongest predictors of conservation impact: MPAs with adequate staff capacity had ecological effects 2.9 times greater than MPAs with inadequate capacity. Thus, continued global expansion of MPAs without adequate investment in human and financial capacity is likely to lead to sub-optimal conservation outcomes