Modified dot-blot hybridization technique for filamentous fungi.

Colony hybridization (Grunstein & Hogness, 1975 Proc. Nat. Acad. Sci. USA 72:3961-3965) A allows the rapid screening of multiple strains for the presence or absence of particular DNA sequences

A rapid and efficient approach for Neurospora crassa transformation using low melting point agarose purified DNA.

A rapid and efficient approach for Neurospora crassa transformation using low melting point agarose purified DNA

Carlene Allen Raper 1925-2019

The field of fungal genetics and biology lost one of its founding anchors with the death of Carlene Raper on September 5, 201

How Temperature Changes Reset a Circadian Oscillator

Circadian rhythms control many physiological activities. The environmental entrainment of rhythms involves the immediate responses of clock components. Levels of the clock protein FRQ were measured in Neurospora at various temperatures; at higher temperatures, the amount of FRQ oscillated around higher levels. Absolute FRQ amounts thus identified different times at different temperatures, so temperature shifts corresponded to shifts in clock time without immediate synthesis or turnover of components. Moderate temperature changes could dominate light-to-dark shifts in the influence of circadian timing. Temperature regulation of clock components could explain temperature resetting of rhythms and how single transitions can initiate rhythmicity from characteristic circadian phases

The Clock affecting 1 mutation of Neurospora is a recurrence of the frq\u3csup\u3e7\u3c/sup\u3e mutation7

The clock affecting-1 (cla-1) mutation of Neurospora crassa increases the period and decreases temperature compensation of the circadian rhythm, and was thought to define an uncloned gene with a possible role in the Neurospora clock. This defect, thought to be due to a translocation, was associated with a slow growth rate and a period of about 27 h at 25cla-1 and found the growth rate and period defects to be due to linked independent mutations. The translocation was not the cause of the long period. The csp-1 mutation, present in the original cla-1 strain, had a period shortening effect, thus cla-1 strains lacking csp-1 had a period length similar to that of frequency7 (frq7). The cla-1 period defect mapped to the frq locus, and sequencing of frq revealed cla-1 to be a re-isolation of frq7

Growth and Structure of Stochastic Sequences

We introduce a class of stochastic integer sequences. In these sequences, every element is a sum of two previous elements, at least one of which is chosen randomly. The interplay between randomness and memory underlying these sequences leads to a wide variety of behaviors ranging from stretched exponential to log-normal to algebraic growth. Interestingly, the set of all possible sequence values has an intricate structure.Comment: 4 pages, 4 figure

A quick RNA mini-prep for Neurospora mycelial cultures

Most RNA isolation techniques currently in use have been developed for the processing of large quantities of material. These typically involve multiple phenol extractions (Reinert et al. 1981 Mol. Cell Biol. 1:829-836) or guanadinium isothio-cyanate/cesium chloride gradients (Chirgwin et al. 1979 Biochem 18:5294-5299) and can be both expensive and time consuming. Often, however, needs arise where quantitatively smaller amounts of RNA are needed from many different samples, for example, during time series analyses or when screening transformants for expression of a transformed gene. Under such circumstances, existing techniques are overly time consuming and yield more RNA than is necessary. The availability of a rapid RNA mini-prep is thus desirable. Such a system has been developed for isolating plant RNA (Nagy et al. 1988 Plant Molecular Biology Manual, B4; ed. Gelvin and Schilperoort, Klewer Academic Publishing, pp. 1-29), and we have adapted this procedure for use with Neurospora and, potentially, other filamentous fungi. Below, we describe the use of this procedure with 50 ml mycelial cultures, although we have used in with equal success with 5 ml cultures without scaling down the amounts of any reagents

Alternative Use of DNA Binding Domains by the Neurospora White Collar Complex Dictates Circadian Regulation and Light Responses

In the Neurospora circadian system, the White Collar complex (WCC) of WC-1 and WC-2 drives transcription of the circadian pacemaker gene frequency (frq), whose gene product, FRQ, as a part of the FRQ-FRH complex (FFC), inhibits its own expression. The WCC is also the principal Neurospora photoreceptor; WCC-mediated light induction of frq resets the clock, and all acute light induction is triggered by WCC binding to promoters of light-induced genes. However, not all acutely light-induced genes are also clock regulated, and conversely, not all clock-regulated direct targets of WCC are light induced; the structural determinants governing the shift from WCC\u27s dark circadian role to its light activation role are poorly described. We report that the DBD region (named for being defective in binding DNA), a basic region in WC-1 proximal to the DNA-binding zinc finger (ZnF) whose function was previously ascribed to nuclear localization, instead plays multiple essential roles assisting in DNA binding and mediating interactions with the FFC. DNA binding for light induction by the WCC requires only WC-2, whereas DNA binding for circadian functions requires WC-2 as well as the ZnF and DBD motif of WC-1. The data suggest a means by which alterations in the tertiary and quaternary structures of the WCC can lead to its distinct functions in the dark and in the light

Development of the CRISPR/Cas9 System for Targeted Gene Disruption in Aspergillus Fumigatus

Low rates of homologous recombination have broadly encumbered genetic studies in the fungal pathogen Aspergillus fumigatus. The CRISPR/Cas9 system of bacteria has recently been developed for targeted mutagenesis of eukaryotic genomes with high effi- ciency and, importantly, through a mechanism independent of homologous repair machinery. As this new technology has not been developed for use in A. fumigatus, we sought to test its feasibility for targeted gene disruption in this organism. As a proof of principle, we first demonstrated that CRISPR/Cas9 can indeed be used for high-efficiency (25 to 53%) targeting of the A. fu- migatus polyketide synthase gene (pksP), as evidenced by the generation of colorless (albino) mutants harboring the expected genomic alteration. We further demonstrated that the constitutive expression of the Cas9 nuclease by itself is not deleterious to A. fumigatus growth or virulence, thus making the CRISPR system compatible with studies involved in pathogenesis. Taken to- gether, these data demonstrate that CRISPR can be utilized for loss-of-function studies in A. fumigatus and has the potential to bolster the genetic toolbox for this important pathogen