The use of Ignite (Basta;glufosinate;phosphinothricin) to select transformants of bar-containing plasmids in Neurospora crassa

The bacterial Basta-resistance (bar) gene has been previously adapted for use as a fungal selectable marker by Avalos et al. (1989 Curr. Genet. 16:369-372) and by Straubinger et al. (1992 Fungal Genet. Newsl. 39:82-83). Both bialaphos and Basta (Ignite) have been developed for use as herbicides and have only been available as free samples from the chemical companies that have been involved in this development

Microwave frequency electromagnetic fields (EMFs) produce widespread neuropsychiatric effects including depression

AbstractNon-thermal microwave/lower frequency electromagnetic fields (EMFs) act via voltage-gated calcium channel (VGCC) activation. Calcium channel blockers block EMF effects and several types of additional evidence confirm this mechanism. Low intensity microwave EMFs have been proposed to produce neuropsychiatric effects, sometimes called microwave syndrome, and the focus of this review is whether these are indeed well documented and consistent with the known mechanism(s) of action of such EMFs. VGCCs occur in very high densities throughout the nervous system and have near universal roles in release of neurotransmitters and neuroendocrine hormones. Soviet and Western literature shows that much of the impact of non-thermal microwave exposures in experimental animals occurs in the brain and peripheral nervous system, such that nervous system histology and function show diverse and substantial changes. These may be generated through roles of VGCC activation, producing excessive neurotransmitter/neuroendocrine release as well as oxidative/nitrosative stress and other responses. Excessive VGCC activity has been shown from genetic polymorphism studies to have roles in producing neuropsychiatric changes in humans. Two U.S. government reports from the 1970s to 1980s provide evidence for many neuropsychiatric effects of non-thermal microwave EMFs, based on occupational exposure studies. 18 more recent epidemiological studies, provide substantial evidence that microwave EMFs from cell/mobile phone base stations, excessive cell/mobile phone usage and from wireless smart meters can each produce similar patterns of neuropsychiatric effects, with several of these studies showing clear dose–response relationships. Lesser evidence from 6 additional studies suggests that short wave, radio station, occupational and digital TV antenna exposures may produce similar neuropsychiatric effects. Among the more commonly reported changes are sleep disturbance/insomnia, headache, depression/depressive symptoms, fatigue/tiredness, dysesthesia, concentration/attention dysfunction, memory changes, dizziness, irritability, loss of appetite/body weight, restlessness/anxiety, nausea, skin burning/tingling/dermographism and EEG changes. In summary, then, the mechanism of action of microwave EMFs, the role of the VGCCs in the brain, the impact of non-thermal EMFs on the brain, extensive epidemiological studies performed over the past 50 years, and five criteria testing for causality, all collectively show that various non-thermal microwave EMF exposures produce diverse neuropsychiatric effects

A lambda/plasmid Cre/lox hybrid vector for large genomic (18kb) fragment insertions and fungal genomic library construction

We have previously constructed lambda/plasmid hybrid vectors designed for both fungal cDNA and genomic library construction (Brunelli and Pall, 1994 Fungal Genet. Newslet. 41:63-65). The genomic library inserts, however, were limited to about 11 kb in size due to the size limitations of lambda packaging. We have constructed a similar vector that has three advantages over these earlier hybrid vectors, as discussed further below. The plasmid pBARGEM7-2 (Pall and Brunelli, 1993. Fungal Genet. Newslet. 40:59-61) was modified by inserting a stuffer sequence into the BamHI site of the polylinker. The stuffer sequence was about 6 kb and can be cut out with BamHI, yielding two BamHI fragments of about 4.5 kb and 1.5 kb. The 4.5 kb fragment contains the lacZ gene, producing very blue colonies (or plaques in lambda) on Xgal medium

New plasmid and lambda/plasmid hybrid vectors and a Neurospora crassa genomic library containing the bar selectable marker and the Cre/lox site-specific recombination system for use in filamentous fungi

In the previous Fungal Genetics Newsletter, we described a series of plasmid vectors constructed carrying the bar gene as a selectable marker for use in filamentous fungi (Pall and Brunelli 1993 Fungal Genetics Newsl. 40:59-63; Pall 1993 Fungal Genetics Newsl. 40:58). In this note, we describe an additional plasmid expression vector carrying this selectable marker and the construction of four llambda/plasmid hybrid vectors carrying the bar gene within plasmid inserts that can excise by Cre/lox-mediated excision. A Neurospora crassa genomic library constructed in one of these lambda/plasmid hybrid vectors is also described below

A series of six compact fungal transformation vectors containing polylinkers with multiple unique restriction sites

In comparison with transformation vectors available for use in E. coli or yeast, there has been relatively little development of vectors for use in filamentous fungi. For example, expression yeast vectors carrying polylinkers flanked by promoters and terminators are available for various uses but such vectors have not been in the public domain for researchers working with filamentous fungi

Transformation of Magnaporthe grisea to phosphinothricin resistance using the bar gene from Streptomyces hygroscopicus

Three transformation systems have been reported for the rice blast fungus Magnaporthe grisea (Parsons et al. 1987 Proc. Natl. Acad. Sci. USA 84:4161-4165; Daboussi et al. 1989 Curr. Genet. 15:453-456; Leung et al. 1990 Curr. Genet. 17:409-411). Among these three selection systems, only hygromycin B resistance provides a dominant selection that can be used for any wild type strain. A second dominant selection marker is needed to transform strains that are already hygromycin B resistant

Homologous recombination following transformation in Neurospora crassa wild type and mutagen sensitive strains

In filamentous fungi transformed with linear DNA, the frequency of gene replacement varies from a few percent in Neurospora crassa, where it depends on the extent of homology between the transforming DNA and the gene to be replaced, (Asch and Kinsey 1990 Mol. Gen. Genet. 22:37-43) to about 30% in Aspergillus (Miller et al. 1985 Mol. Cell Biol. 5:17-14). Yet, in the yeast, Saccharomyces cerevisiae, homologous gene replacement is the rule and ectopic integration of transforming DNA is extremely rare (Fincham 1989 Microbiol. Rev. 53:148-170). This variation could be the result of the action of one or a few genetic pathways and thus, be affected by single mutations. Mutants with altered ability to integrate transforming DNA may be sensitive to ionizing radiation, since double strand break repair is necessary for both recombination and survival after ionizing radiation damage. Therefore, we looked among the radiation-sensitive mutants, uvs-2,3,6, mus-9,11, and mei-2,3, for changes in the ability to integrate transforming DNA either ectopically or by homologous recombination. All mutant strains were extensively backcrossed to wild type strains of 74-OR23-1A background and the 74-OR23-1A strain served as a control

Mechanistic insight into RET kinase inhibitors targeting the DFG-out conformation in RET-rearranged cancer

Oncogenic fusion events have been identified in a broad range of tumors. Among them, RET rearrangements represent distinct and potentially druggable targets that are recurrently found in lung adenocarcinomas. Here, we provide further evidence that current anti-RET drugs may not be potent enough to induce durable responses in such tumors. We report that potent inhibitors such as AD80 or ponatinib that stably bind in the DFG-out conformation of RET may overcome these limitations and selectively kill RET-rearranged tumors. Using chemical genomics in conjunction with phosphoproteomic analyses in RET-rearranged cells we identify the CCDC6-RETI788N mutation and drug-induced MAPK pathway reactivation as possible mechanisms, by which tumors may escape the activity of RET inhibitors. Our data provide mechanistic insight into the druggability of RET kinase fusions that may be of help for the development of effective therapies targeting such tumors

Exome-wide Rare Variant Analysis Identifies TUBA4A Mutations Associated with Familial ALS

Exome sequencing is an effective strategy for identifying human disease genes. However, this methodology is difficult in late-onset diseases where limited availability of DNA from informative family members prohibits comprehensive segregation analysis. To overcome this limitation, we performed an exome-wide rare variant burden analysis of 363 index cases with familial ALS (FALS). The results revealed an excess of patient variants within TUBA4A, the gene encoding the Tubulin, Alpha 4A protein. Analysis of a further 272 FALS cases and 5,510 internal controls confirmed the overrepresentation as statistically significant and replicable. Functional analyses revealed that TUBA4A mutants destabilize the microtubule network, diminishing its repolymerization capability. These results further emphasize the role of cytoskeletal defects in ALS and demonstrate the power of gene-based rare variant analyses in situations where causal genes cannot be identified through traditional segregation analysis