Perspectives on genetic resources at the Fungal Genetics Stock Center.

The Fungal Genetics Stock Center has been banking and distributing resources for work with genetically characterized fungi since 1960. While most of the collection consists of strains of Neurospora, an NIH model filamentous fungus, the past fifteen years has seen the collection expand to include plant and human pathogenic fungi. The use of the resources in the collection has grown over the last 10 years as well, reflecting both a growth in research using standardized materials as well as the development of new materials through molecular genetic technology. This growth is not limited to newly deposited materials, however, and includes renewed interest in particular classes of strains with characteristics that were not recognized when they were originally deposited. One significant example is the use of strains carrying the osmotic-2 lesion in Neurospora crassa. This, and other utilization trends, underscores the need to provide strong support to the continued and expanded biobanking effort in the US

Fungal Genetics Stock Center Catalogue of Strains, 10th Edition

Catalogue of Strains, 10th edition, 2004, supplement to Fungal Genetics Newsletter No. 51. This catalogue contains lists of materials held by the Fungal Genetics Stock Center

Work in the dark to harvest large liquid-grown cultures

Biochemical purification of low-abundance proteins from Neurospora crassa often requires collection of \u3e100 g wet weight of mycelial mass. For purification of the dynein motor from N. crassa, 4 to 8 one liter liquid cultures are inoculated with 1 x 106 conidia/ml at 3:00 pm and incubate overnight at 28°C with shaking. At 9:00 am the next morning, mycelia (10- 15 g/flask) are collected by filtration using a new cellulose filter for each flask (Fisherbrand P8). Unfortunately, we frequently find that mycelia are easily collected from the first one to three flasks, however, mycelia cannot be harvested from the remaining flasks because the filters become clogged. We have determined that this is a light-dependent phenomenon. If the incubators are covered in black trash bags for the overnight incubation and the lab lights are not turned on during the morning harvesting period, we no longer see any clogging of filters. We suspect that light-induction of hydrophobins is the cause of the clogging of cellulose filters (Lauter et al. 1992)

Demonstration that the Neurospora crassa mutation un-4 is a single nucleotide change in the tim16 gene encoding a subunit of the mitochondrial inner membrane translocase

The Neurospora crassa temperature sensitive mutation known as un-4 has been shown by a map-based complementation approach to be a single nucleotide change in the open reading frame of the mitochondrial inner membrane translocase subunit tim16 (NCU05515)

Fantastic growth as the FGSC turns 50

Comparative Medicine - OneHealth and Comparative Medicine Poster SessionFounded in 1960, the Fungal Genetics Stock Center enters it's fiftieth year of operation during a period of tremendous growth. The collection has more than doubled since moving to UMKC in 2004 and has added new materials that reach out beyond it's traditional constituency. Among these are deletion sets for Neurospora, Cryptococcus and Candida as well as molecular genetic tools for working with industrial fungi, model organisms, and plant and human pathogens. With distribution growing every year, the FGSC sends materials to scientists in over 35 countries every year; approximately half of our orders are from within the US. In addition to being part of an NIH funded multi-institution Functional Genomics Program for Neurospora, the FGSC is involved in cutting edge genomics research with collaborators at the US DOE Joint Genome Institute. The FGSC and its staff are actively involved in national and international societies and ad hoc working groups fostering the development of collection resources in the US and around the world

Kinesin and dynein mutants provide novel insights into the roles of vesicle traffic during cell morphogenesis in Neurospora

AbstractBackground: Kinesin and cytoplasmic dynein are force-generating molecules that move in opposite directions along microtubules. They have been implicated in the directed transport of a wide variety of cellular organelles, but it is unclear whether they have overlapping or largely independent functions.Results: We analyzed organelle transport in kinesin and dynein single mutants, and in a kinesin and dynein double mutant of Neurospora crassa. Remarkably, the simultaneous mutation of kinesin and dynein was not lethal and resulted in an additive phenotype that combined the features of the single mutants. The mutation of kinesin and dynein had opposite effects on the apical and retrograde transport, respectively, of vesicular organelles. In the kinesin mutant, apical movement of submicroscopic, secretory vesicles to the Spitzenkörper – an organelle in the hyphal apex – was defective, whereas the predominantly retrograde movement of microscopic organelles was only slightly reduced. In contrast, the dynein mutant still had a prominent Spitzenkörper, demonstrating that apical transport was intact, but retrograde transport was essentially inhibited completely. A major defect in vacuole formation and dynamics was also evident. In agreement with the observations on apical transport, protein secretion into the medium was markedly inhibited in the kinesin mutant but not in the dynein mutant.Conclusions: Transport of secretory vesicles is necessary but not sufficient for normal apical extension. A component of retrograde transport, presumably precursors of the vacuole system, is also essential. Our findings provide new information on the role microtubule motors play in cell morphogenesis and suggest that kinesin and cytoplasmic dynein have largely independent functions within separate pathways

Identification of the Neurospora crassa mutation un-10 as a point mutation in a gene encoding eukaryotic translation initiation factor 3, subunit B.

The Neurospora crassa temperature-sensitive mutant known as un-10 has been shown by a map-based complementation approach to be a single nucleotide change in the open reading frame of the eukaryotic translation initiation factor 3b (NCU02208.3)

Complementation of un-16 and the development of a selectable marker for transformation of Neurospora crassa

Although nearly sixty temperature-sensitive lesions have been mapped in Neurospora crassa, most of their functions have not been identified. These loci are called unknown (un). As part of an effort to identify the open reading frame associated with one of these, we undertook to walk to un-16 using the complementation of temperature-sensitivity as a selection. Cosmids complementing un-16 were identified and the un-16 gene was subcloned. DNA sequence analysis of un-16 revealed that it encodes the highly conserved S9 protein of the 40S ribosomal subunit. This gene has proven useful as a selectable marker and may provide a simple mechanism for the controlled alteration of protein synthesis in N. crassa

Experimental evaluation of interfaces using atomic-resolution high angle annular dark field (HAADF) imaging

Aberration-corrected highangleannulardarkfield (HAADF) imaging in scanning transmission electron microscopy (STEM) can now be performed at atomic-resolution. This is an important tool for the characterisation of the latest semiconductor devices that require individual layers to be grown to an accuracy of a few atomic layers. However, the actual quantification of interfacial sharpness at the atomic-scale can be a complicated matter. For instance, it is not clear how the use of the total, atomic column or background HAADF signals can affect the measured sharpness or individual layer widths. Moreover, a reliable and consistent method of measurement is necessary. To highlight these issues, two types of AlAs/GaAs interfaces were studied in-depth by atomic-resolutionHAADFimaging. A method of analysis was developed in order to map the various HAADF signals across an image and to reliably determine interfacial sharpness. The results demonstrated that the level of perceived interfacial sharpness can vary significantly with specimen thickness and the choice of HAADF signal. Individual layer widths were also shown to have some dependence on the choice of HAADF signal. Hence, it is crucial to have an awareness of which part of the HAADF signal is chosen for analysis along with possible specimen thickness effects for future HAADF studies performed at the scale of a few atomic layers

Characterization of the Temperature-Sensitive Mutations un-7 and png-1 in Neurospora crassa

The model filamentous fungus Neurospora crassa has been studied for over fifty years and many temperature-sensitive mutants have been generated. While most of these have been mapped genetically, many remain anonymous. The mutation in the N. crassa temperature-sensitive lethal mutant un-7 was identified by a complementation based approach as being in the open reading frame designated NCU00651 on linkage group I. Other mutations in this gene have been identified that lead to a temperature-sensitive morphological phenotype called png-1. The mutations underlying un-7 result in a serine to phenylalanine change at position 273 and an isoleucine to valine change at position 390, while the mutation in png-1 was found to result in a serine to leucine change at position 279 although there were other conservative changes in this allele. The overall morphology of the strain carrying the un-7 mutation is compared to strains carrying the png-1 mutation and these mutations are evaluated in the context of other temperature-sensitive mutants in Neurospora