Meiotic Recombination in Neurospora crassa Proceeds by Two Pathways with Extensive Holliday Junction Migration

Analysis of thousands of Δmsh-2 octads using our fluorescent recombination system indicates that, as in other filamentous fungi, symmetric heteroduplex is common in the his-3 region of Neurospora crassa. Symmetric heteroduplex arises from Holliday junction migration, and we suggest this mechanism explains the high frequency of His⁺ spores in heteroallelic crosses in which recombination is initiated cis to the his-3 allele further from the initiator, cog⁺. In contrast, when recombination is initiated cis to the his-3 allele closer to cog⁺, His⁺ spores are mainly a result of synthesis-dependent strand annealing, yielding asymmetric heteroduplex. Loss of Msh-2 function increases measures of allelic recombination in both his-3 and the fluorescent marker gene, indicating that mismatches in asymmetric heteroduplex, as in Saccharomyces cerevisiae, tend to be repaired in the direction of restoration. Furthermore, the presence of substantial numbers of conversion octads in crosses lacking Msh-2 function suggests that the disjunction pathway described in S. cerevisiae is also active in Neurospora, adding to evidence for a universal model for meiotic recombination.     

The mechanism of uptake of cobalt ions by Neurospora crassa

Uptake of Co(2+) by 3-day-old mycelia of Neurospora crassa involves cell-surface binding as well as transport into the intracellular space. The surface binding is rapid and accounts for 30-40% of the total Co(2+) uptake. Transport of Co(2+) occurs at a rate of 40mug/h per 100mg dry wt. Surface binding and overall uptake show different temperature dependence. Metabolic inhibitors such as azide, dinitrophenol and fluoride depress transport of Co(2+). The overall uptake of Co(2+) exhibits a high K(m) value and hence the concentration mechanism is one of low ;affinity' for the metal. The uptake of Co(2+) varies linearly with pH in the range pH3 to pH6. Mg(2+) inhibits both surface binding and transport of Co(2+). It is suggested that the system that transports Mg(2+) is also involved in Co(2+) uptake by N. crassa.     

Some required events in conidial germination of Neurospora crassa.

A temperature-sensitive mutant of Neurospora crassa, with reduced levels of protein synthesis at 37°C, was used to identify some essential events in conidial germination. Conidia of mutant strain psi-1 were incubated for 2 hr at 37°C and then shifted to 20°C. Germination was inhibited at 37°C, but commenced after 1.5 hr at 20°C. Increases in aspartate transcarbamylase activity, cell wall synthesis, and nuclear number preceded germination. However, increases in glutamate dehydrogenase activity, amino acid uptake, and DNA synthesis were inhibited prior to germination. Although all of these events were correlated with germination in control cultures of the mutant at 20°C and of its parent strain at 20 and 37°C, some events were apparently not essential for germination. The requirement for aspartate transcarbamylase activity was demonstrated independently by the failure of strain pyr-3d (lacking the activity) to germinate in the absence of uridine. The dispensability of glutamate dehydrogenase activity and DNA synthesis for the germination of some conidia was verified by the germination of strain am-1 (lacking glutamate dehydrogenase activity) in the absence of glutamate and by the germination of the parent strain in the presence of hydroxyurea (an inhibitor of DNA synthesis). These findings identify some landmarks in germination which may be useful in further studies of the regulation of a developmental program. They also provide preliminary evidence that the resting conidia may contain nuclei arrested at different stages of their division cycle.     

Photoperiodism in Neurospora crassa

Plants and animals use day or night length for seasonal control of reproduction and other biological functions. Overwhelming evidence suggests that this photoperiodic mechanism relies on a functional circadian system. Recent progress has defined how flowering time in plants is regulated by photoperiodic control of output pathways, but the underlying mechanisms of photoperiodism remain to be described. The authors investigate photoperiodism in a genetic model system for circadian rhythms research, Neurospora crassa. They find that both propagation and reproduction respond systematically to photoperiod. Furthermore, a nonreproductive light-regulated function is also enhanced under certain photoperiodic conditions. All of these photoperiodic responses require a functional circadian clock, in that they are absent in a clock mutant. Night break experiments show that measuring night length is one of the mechanisms used for photoperiod assessment. This represents the first formal report of photoperiodism in the fungi.     

Cellular events in growth and germination of giant conidia of Neurospora crassa

During growth of conidia in 3.22 M ethylene glycol the increase in the number of the nuclei is proportional to the increase in volume only in the phase of maximum growth rate and is lower in the preceding and in the following periods of growth. DNA synthesis similarly initiates later and decreases faster than protein synthesis. The dilution of ethylene glycol is followed by the germination of giant conidia, which is characterized by the absence of a lag phase, a high degree of synchrony and the formation of more than one germ tube per conidium. The number of germ tubes is dependent on the volume reached by conidia at the end of the treatment and does not increase with time. The resuming of DNA synthesis after germination is preceded by a sharp increase in protein synthesis and the division of almost half of the nuclei and shows a synchronized pattern. Results are discussed in the light of models of growth proposed for eukaryotic cells.     

Metabolism of ricinoleate by Neurospora crassa

Neurospora crassa is a potential expression system for evaluating fatty-acid-modifying genes from plants producing uncommon fatty acids. One such gene encodes the hydroxylase that converts oleate to ricinoleate, a fatty acid with important industrial uses. To develop this expression system, it is critical to evaluate the metabolism and physiological effects of the expected novel fatty acid(s). We therefore examined effects of ricinoleate on lipid biosynthesis and growth of N. crassa. Ricinoleate inhibited growth and reduced levels of phospholipids and of 2-hydroxy fatty acids in glycolipids, but led to increased lipid accumulation on a mass basis. To evaluate incorporation and metabolism of ricinoleate, we followed the fate of 14 M–3 mM [1-¹⁴C]ricinoleate. The fate of the [¹⁴C]ricinoleate was concentration-dependent. At higher concentrations, ricinoleate was principally incorporated into triacylglycerols. At lower concentrations, ricinoleate was principally metabolized to other compounds. Thus, N. crassa transformants expressing the hydroxylase gene can be detected if the level of hydroxylase expression allows both growth and ricinoleate accumulation.     

Uptake of Nitrite by Neurospora crassa

Like the nitrate transport system, the nitrite uptake system in Neurospora crassa is induced by either nitrate or nitrite. This induction is prevented by cycloheximide, puromycin, or 6-methyl purine. The K(m) for nitrite of the induced nitrite uptake system is 86 muM, and the V(max) is 100 mumol of nitrite per g (wet weight) per h. Nitrite uptake is inhibited by metabolic poisons such as arsenate, dinitrophenol, cyanide, and antimycin A. No repression or inhibition of the nitrite transport system by ammonia, nitrate, or Casamino Acids was observed.     

Peptide utilization by nitrogen-starved Neurospora crassa

Peptides ranging in size from a mean number of 30 residues down to dipeptides supported growth of a leucine auxotroph when used as both a nitrogen and leucine source. Under nitrogen-limiting conditions, the peptides induced extracellular peptidohydrolytic activity, hydrolyzing peptides to monomer amino acids. Growth of a leu-2 mutant of Neurospora crassa on those peptides transportable by the oligopeptide transport system did not result in induction of hydrolytic activity, whereas growth of a leu-2; gltR mutant on these same peptides resulted in induction of peptidohydrolytic activity. The induced extracellular proteolytic activity was shown to be analogous to that inducible by growth on proteins, e.g., bovine serum albumin.     

Karyotyping of Neurospora crassa using synaptonemal complex spreads of translocation quadrivalents.

The purposes of the present research are (i) to establish the karyotype of Neursopora crassa using visualization of kinetochores in the synaptonemal complex (SC) spreads, (ii) to assign each chromosome to a linkage group, and (iii) to examine chromosome pairing and recombination nodules in quadrivalents. Two strains containing reciprocal translocations were used: T(I;II)4637, which involves linkage groups I and II, and alcoy, which contains 3 independent translocations involving I and II, IV and V, and III and VI. Visualization of kinetochores in the spreads requires the use of freshly prepared fixatives. Kinetochore locations and arm ratios were documented in all 7 N. crassa chromosomes. This new information, based on kinetochore position, arm ratios, chromosome length, and quadrivalent analyses, enabled unequivocal confirmation of chromosome assignments to genetic linkage groups. Chromosome pairing in a translocation quadrivalent starts at the 4 terminal regions, and proceeds right up to the translocation break point. Recombination nodules are found in all 4 arms of quadrivalents. The ability to identify a specific chromosome to a genetic linkage group together with the ability to visualize recombination nodules and their locations will allow future cytological analysis of recombination events.     

Expression of a plant protein by Neurospora crassa.

Heterologous expression of plant genes may serve as an important alternative for producing plant proteins. We have investigated the ability of the fungus Neurospora crassa to secrete zeamatin, a protein produced by Zea mays. Zeamatin was induced after being fused to glucoamylase, an extracellular hydrolase produced by N. crassa. Glucoamylase induction and other culture parameters were monitored in untransformed N. crassa grown in shaken liquid culture. A DNA plasmid, pGEZ, was constructed by inserting zeamatin-encoding cDNA into an expression cassette containing the promoter, a truncated open reading frame, and the terminator sequence of the N. crassa glucoamylase gene. Zeamatin-encoding cDNA was modified at the N terminus to include a kex-2 protease site, allowing cleavage of the chimeric product in the secretory pathway. Strains containing the chimeric gene construct were grown in liquid culture and induced for glucoamylase and zeamatin production. Zeamatin antibody detected a protein in a Western blot of concentrated culture supernatants that comigrated with authentic zeamatin. Secreted zeamatin was active in inhibiting the growth of Candida albicans in an agar diffusion assay, indicating that zeamatin had been correctly synthesized, processed, and secreted by N. crassa.     

Gene disruption by transformation in Neurospora crassa.

To establish conditions which might permit deliberate gene disruptions in Neurospora crassa, we studied transformation with linear DNA fragments. The transformation frequency observed was increased about twofold in comparison with that obtained with circular plasmid DNA. However, only a low proportion, approximately 10%, of the integration events occurred at the homologous site, whereas most integrations of transforming DNA took place in nonhomologous regions. It was also found that multiple integration events frequently occurred in individual transformants. A plasmid, designated pJP12, was constructed that contains the N. crassa am+ gene interrupted by insertion into its coding region of a DNA segment carrying a functional Neurospora qa-2+ gene. A fragment of Neurospora DNA that contains this am qa-2+ construction was obtained from plasmid pJP12 and used to transform an am+ qa-2 strain in an attempt to disrupt the resident am+ gene. After the initial qa-2+ transformants were converted to homokaryons by appropriate crosses, 10 independent transformants with an am mutant phenotype were found among 117 examined. Each of these qa-2+ am transformants showed the loss of a hybridization band in Southern blots of genomic DNA that corresponded to the normal am+ gene and the presence of a new hybridization band, consistent with an alteration in the am+ region.     

Protease secretion in Neurospora crassa

Secreted and constitutive intracellular proteases of $\text{Neurospora crassa}$ differ with regard to inhibitor sensitivity, substrate specificity, isoelectric points and other properties. Upon the induction of protease secretion the enzymes released from the mycelium are formed $\text{de novo}$ as demonstrated by density labelling with D₂O. Vesicles which contain the constitutive intracellular proteases are, therefore, not involved in the secretion of proteases.     

Postreplication repair in Neurospora crassa

Summary Changes in the molecular weight of nascent DNA made after ultraviolet (UV) irradiation have been studied in the excision-defective Neurospora mutant uvs-2 using isotopic pulse labeling, alkaline gradient centrifugation and alkaline filter elution. Both the size of nascent DNA and the rate of incorporation of label into DNA was reduced by UV light in a dose dependent manner. However, this DNA repair mutant did recover the ability to synthesize control-like high molecular weight DNA 3 hours after UV treatment, although the rate of DNA synthesis remained depressed after the temporary block to elongation (or ligation) had been overcome. Photoreactivation partially eliminated the depression of DNA synthesis rate and UV light killing of cells, providing strong evidence that the effects on DNA synthesis and killing were caused by pyrimidine cyclobutane dimers. The caffeine inhibition repair studies performed were difficult to quantitate but did suggest either partial inhibition of a single repair pathway or alternate postreplication DNA repair pathways in Neurospora. No enhancement in killing was detected after UV irradiation when cells were grown on caffeine containing plates.