Effects of in vitro and in vivo supplementation with zinc on superoxide anion production in leukocytes

The effects of zinc on the rate of production of bactericidal O2- of polymorphonuclear leukocytes (PMN) in response to three different types of stimulating agents (serum-treated zymosan (STZ), Con A, and myristate) were studied. The percentage reduction of O2- production of PMN stimulated by STZ, Con A, and myristate were all reduced in response to Zn, irregardless of whether Zn was added to the reaction mixture immediately before SZT addition or following a prior 20 min. incubation of PMN in the presence of Zn. However, when Zn was introduced intraperitonially into guinea pigs before the collection of PMN from the animal, zinc treatment produced inhibition only in STZ-activated PMN; it produced no effect in O2- production of PMN stimulated by myristate, and it further augmented the O2- production stimulated by Con A.     

Granulocyte-macrophage colony-stimulating factor is a major macrophage fusion factor present in conditioned medium of concanavalin A-stimulated spleen cell cultures

In 1983, we reported that the conditioned medium (CM) of spleen cell cultures treated with Con A greatly induced fusion of mouse alveolar macrophages within 2 to 3 days at a very high rate of more than 80% (Proc. Natl. Acad. Sci. USA 80:5583, 1983). In the course of examining macrophage fusion factors (MFF) present in Con A-CM, we found that IL-4 induced fusion of alveolar macrophages with a time course similar to that induced by Con A-CM. However, the maximal fusion rate induced by IL-4 (4 ng/ml) was about 35%. Furthermore, the fusion induced by Con A-CM was blocked only partially by adding IL-4 antibody, indicating that there are unknown MFF other than in Con A-CM. Of several other cytokines produced by Con A-stimulated spleen cells, IL-6 (20 ng/ml), IFN-gamma (45 ng/ml) and granulocyte-macrophage (GM)-CSF (10 ng/ml) greatly potentiated the fusion induced by 4 ng/ml of IL-4. The assay of these cytokines in Con A-CM proved that it contained 0.44 +/- 0.04 ng/ml of IL-4, 1.0 +/- 0.24 ng/ml of IL-6, 9.1 +/- 0.07 ng/ml of IFN-gamma, and 11.6 +/- 1.66 ng/ml of GM-CSF. When the potentiating effects of IL-6, IFN-gamma and GM-CSF on macrophage fusion were examined in the presence of 0.4 ng/ml of IL-4, only GM-CSF increased the fusion rate to the maximal level induced by Con A-CM at its physiologic concentration (10 ng/ml). The macrophage fusion induced by Con A-CM was greatly suppressed by adding antibody against GM-CSF. GM-CSF had a biphasic effect on growth and fusion, depending on its dose levels used: 0.01 to 0.1 ng/ml increased proliferation without inducing fusion and 10 ng/ml preferentially induced fusion. There was a negative relationship between macrophage growth and fusion. IL-4 was a potent inhibitor of proliferation of macrophages induced by GM-CSF. These results clearly indicate that GM-CSF is a major MFF present in Con A-CM.     

Selection and characterization of Chinese hamster ovary cells resistant to the cytotoxicity of lectins.

Chinese hamster ovary (CHO) cells selected in a single step for resistance to the cytotoxicity of the lectin from red kidney beans (PHA) behave as authentic somatic cell mutants. The PHA-resistant (Phar) phenotype is stable in the absence of selection; its frequency in a sensitive-population is increased several-fold by mutagenesis; and it behaves recessively in somatic cell hybrids. The activity of a specific glycosyl transferase which transfers N-acetylglucosamine (GlcNAc) to terminal alpha-mannose residues is dramatically reduced (less than or equal to 5% of the activity detected in wild-type CHO cells) in several independent PhaR clones. These clones also exhibit (a) a decreased ability to bind [125I]-PHA; (b) a marked resistance to the cytotoxicity of wheat germ agglutinin (WGA), Ricin (RIC) and Lens culinaris agglutinin (LCA); (c) a 4- to 5-fold increased sensitivity to the cytoxocity of concanavalin A (Con A); (d) an increased ability to bind 125I-Con A; and (e) decreased surface galactose residues - all properties consistent with the specific loss of the GlcNAc transferase activity. The lectins WGA, RIC, LCA and Con A have also been used to select, in a single step, resistance closes from each of two complementary CHO auxitrophic lines. These lectin-resistant clones have been characterized by their ability to survive cytotoxic doses of PHA, Con A, WGA, RIC, or LCA, and 4-5 "lectin-resistance" phenotypes have been demonstrated. Complementation data is being sought by somatic cell hybridization. Preliminary results show that two phenotypically-distinct Con AR mutants are complementary in that hybrid cells formed between them exhibit wild-type sensitivity to Con A.     

Mutagenicity and clastogenicity of proflavin in L5178Y/TK +/- -3.7.2.C cells

We evaluated the ability of proflavin to induce specific-locus mutations at the heterozygous thymidine kinase (tk) locus of L5178Y/TK +/- -3.7.2C mouse lymphoma cells, which appears to permit the recovery of mutants due to single-gene and chromosomal mutations. Proflavin was highly mutagenic at the tk locus, producing 724-965 TK mutants/10(6) survivors (background = 56-85/10(6); survival = 29-32%). Most of the mutants were small colonies, which suggested that proflavin may induce chromosomal mutations. The potent clastogenicity of proflavin was confirmed by cytogenetic analysis for chromosomal aberrations. At the highest dose analyzed (1.5 micrograms/ml), proflavin produced 82 aberrations/100 metaphaes (background = 2/100). The large-colony TK mutant frequency produced by proflavin (48-109/10(6) survivors; background = 23/10(6); survival = 57-61%) was similar to published HPRT mutant frequencies produces by proflavin in L5178Y and CHO cells (50-100/10(6) survivors; background = 2-50/10(6); survival = 50-62%). These results lead to the conclusion that proflavin is a potent clastogen and induces a high frequency of small-colony TK mutants; however, it induces a low frequency of HPRT mutants and a low frequency of large-colony TK mutants.     

Genotoxicity of 2-amino-6-N-hydroxyadenine (AHA) to mouse lymphoma and CHO cells.

2-Amino-6-N-hydroxyadenine (AHA) treated L5178Y/TK (+/-)-3.7.2C mouse lymphoma cells were evaluated for mutations at the tk, hgprt, and Na+/K+ ATPase loci, as well as for gross chromosome aberrations and induction of micronuclei. In addition, AHA was evaluated for its ability to induce HGPRT mutants in CHO cells. AHA was found to induce mutations at all evaluated loci and in both cell types. The TK mutants were primarily large colonies although a few small colonies were also induced, particularly at the higher concentrations. Preliminary cytogenetic analysis of AHA-treated mouse lymphoma cells indicated that some gross aberrations but not micronuclei were induced. The 20 small-colony TK mutants evaluated by banded karyotype indicate that only a small fraction (2 of 20) showed chromosome 11 abnormalities. From these studies, it appears that AHA may be one of a very few chemicals that is capable of inducing multi-locus point mutations, with only slight clastogenic activity. Particularly at the higher concentrations, some of the mutants may contain multi-locus point mutations that result in slow growth.     

Synergism of mutant frequencies in the mouse lymphoma cell mutagenicity assay by binary mixtures of methyl methanesulfonate and ethyl methanesulfonate

The effect of mixed mutagen exposures on the rate and type of induced mutants was studied in the L5178Y/TK+/-----TK-/- mouse lymphoma cell mutagenicity assay. In this assay, exposure to ethyl methanesulfonate (EMS) results in more mutants that form large colonies than small colonies. Exposure to methyl methanesulfonate (MMS) results in more mutants that form small colonies than large colonies. Other reports in the literature suggest that large colony TK-/- mutants appear to result from small-scale, perhaps single-gene mutations, and that small-colony TK-/- mutants appear to be associated with chromosomal mutations. Treating cells for 4 h with simple, 2-component mixtures containing 6.45 micrograms/ml MMS and either 261, 392, 560 or 712 micrograms/ml EMS resulted in synergism of mutants at each mixture level. The frequencies of total mutants were synergized 12, 20, 35 and 72%, respectively, in mixed exposures with graded doses of EMS, above the sums of the mixture components. Small colony mutants were synergized to a greater extent than large colony mutants. The frequencies of small colony mutants in mixed exposures were increased 31, 54, 73 and 123%, respectively, while the frequencies of large colony mutants were increased -7, -6, 11 and 39%. Statistical analyses provide strong evidence of synergism (within the limits of the assay) for total and small-colony mutants at all doses of EMS tested, and for large-colony mutants above 400 micrograms/ml EMS. Similar magnitudes of synergism resulted when other constant levels of MMS (4.30 or 8.60 micrograms/ml) were mixed with the same graded doses of EMS. The degree of synergism was dependent on EMS concentration but not on MMS concentration.     

N-methyl-N'-nitro-N-nitrosoguanidine and UV induced mutants of the dinoflagellate Crypthecodinium cohnii

Mutant strains were chemically induced by treatment with N-methyl-N'-nitro-N-nitrosoguanidine (NTG) and UV irradiation. UV and NTG mutation rates were obtained that were both consistent with the organism being haploid. Three types of mutants were produced: (a) strains deficient in both beta- and gamma-carotene, the only carotenoids found in the wild type; phenotypes include albinos (translucent, dull white, "snow white") and cream-colored on agar as compared to the yellow-orange color of wild type colonies; (b) strains requiring adenine, guanine or cytosine in addition to the minimal medium for growth; (c) mutants that grow at a rate less than 40% of the wild type in minimal medium.     

Spontaneous Mutation Rates in Mammalian Cells: Effect of Differential Growth Rates and Phenotypic Lag

We calculated a spontaneous rate of 26-37 x 10(-6) mutations per cell division for L5178Y MOLY (mouse lymphoma) cells at the thymidine kinase locus (tk+/(-)----tk-/-) using a procedure that isolated and segregated cells during expression. This rate was 50 times higher than when cells expressed the mutant phenotype in suspension. The higher mutation rates obtained with the in situ procedure suggest that many of the mutants, whether expressed or unexpressed, grew more slowly than wild-type cells prior to selection with trifluorothymidine (TFT), implying that the slow growth phenotype is expressed earlier than the TFTr (TFT-resistant) phenotype. The loss of mutants was not restricted to cells forming small colonies; the mutation rate for cells forming large colonies was more than ten times higher using the in situ procedure. In this new procedure, the cells expressed spontaneous mutations while growing in semisolid medium for up to 3 days without TFT. Mutants were then selected in situ by adding an overlay of TFT and the visible colonies were analyzed after 11 days. Cells with spontaneous mutations at the tk locus required approximately 30 hr for the more rapidly expressing cells with new mutations to be detected. Most of the TFTr colonies selected after 60 hr of growth in semisolid medium represented independent mutations that had accumulated during the first 30 hr.(ABSTRACT TRUNCATED AT 250 WORDS)     

N-methyl-N′-nitro-N-nitrosoguanidine and UV induced mutants of the dinoflagellate Crypthecodinium cohnii*

SYNOPSIS. Mutant strains were chemically induced by treatment with N-methyl-N′-nitro-N-nitrosoguanidine (NTG) and UV irradiation. UV and NTG mutation rates were obtained that were both consistent with the organism being haploid. Three types of mutants were produced: (a) strains deficient in both β- and γ-carotene, the only carotenoids found in the wild type; phenotypes include albinos (translucent, dull white, “snow white”) and cream-colored on agar as compared to the yellow-orange color of wild type colonies; (b) strains requiring adenine, guanine or cytosine in addition to the minimal medium for growth; (c) mutants that grow at a rate less than 40% of the wild type in minimal medium.     

Isolation and genetic analysis of nuclease halo (nuh) mutants of Neurospora crassa.

Summary Nuclease halo (nuh) mutants of the ascomycete Neurospora crassa have been isolated which are characterized reduced release of deoxyribonuclease (DNase) activities from colonies grown on sorbose-containing agar media. To identify nuh mutants, mutagenized isolates were transferred to commercial DNase test agar, or grown on minimal medium and then overlayed with agar that contained heat-denatured DNA. DNase activity was visualized by acid precipitation which produced clear rings of digestion (haloes) around the colonies.To identify the number of genes in which mutations lead to reduced release of nuclease activity, eleven nuh mutants were checked for close linkage and linked pairs were tested for complementation. These mutants were assigned to eight genes, and all except one were mapped in six small regions of the Neurospora linkage maps. In addition, among a large number of existing mutants which were tested for nuclease haloes, two mutants were found that showed the Nuh phenotype, namely uvs-3 and uvs-6. One of the isolated nuh mutants was also found to be sensitive to UV and was mapped close to uvs-3; it may represent a new allele of this gene.As a first step towards identification of genuine nuclease mutants, extensively backcrossed strains of mutants from different genes have been assayed for nuclease activity with denatured DNA in extracts. A pronounced reduction, compared to wild type at the same stage of growth, was found in uvs-3 and also in nuh-3, a mutant that is not UV-sensitive.     

Lectin binding to newt epidermis: fluorescent localization and effects on motility

The ability of seven lectins to bind to newt epidermal cells and influence their motility was examined. Of the seven fluoresceinated lectins applied to frozen sections containing intact newt skin and migrating epidermis (wound epithelium), only Con A (concanavalin A), WGA (wheat germ agglutinin), and PNA (peanut agglutinin) produced detectable epidermal fluorescence. Con A and WGA each heavily labeled all layers of intact epidermis, but PNA bound only to the more superficial layers. In contrast to a single population of labeled cells in migrating epidermal sheets after treatment with Con A, there were both labeled and unlabeled cells after exposure to either WGA or PNA. The wound bed was labeled by both Con A and WGA, but not by PNA. DBA (Dolichos bifloris agglutinin), RCA I (Ricinus communis agglutinin), and UEA (Ulex europaeus agglutinin), did not produce significant fluorescence with either migrating or intact epidermis. In general, inhibitory effects on epidermal motility correlated with the binding studies. Thus, Con A, WGA, and PNA, the lectins which clearly bound to the epidermis, all produced a concentration-dependent depression in the rate of epidermal wound closure. RCA was somewhat paradoxical in that it was moderately inhibitory despite showing essentially no binding. The effects of SBA and UEA were equivocal. DBA had no effect. These results indicate that the inhibition of motility produced by Con A that we have described previously is not peculiar to this mannose-binding lectin, but is shared by at least one lectin with an affinity for D-GlcNAc (WGA), and one with an affinity for B-D-Gal(1-3)-D-GalNAc (PNA).(ABSTRACT TRUNCATED AT 250 WORDS)     

Alloreactive T cells from individual soft agar colonies specific for guinea pig Ia antigens. II. Cellular and molecular requirements for optimal proliferative responses

We have investigated the cellular and molecular requirement for optimal proliferative responses of several alloreactive T cell lines that were derived from individual soft agar colonies and were specific for guinea pig Ia antigens. Optimal proliferation of several colonies was observed in cultures containing purified allogeneic macrophages and growth factor(s) present in supernatant fluids of Con A-activated T cells (Con A-S). Significant proliferative responses of these alloreactive T cell colonies were also routinely detected in cultures only supplemented with unfractionated irradiated allogeneic peritoneal exudate cell (PEC). The T cell component of the stimulator cell population was crucial for these responses by producing necessary growth factor(s) endogenously in the culture. Thus, 2 signals, allogeneic Ia antigens and growth factor(s), were required for optimal proliferative responses of these alloreactive T cell colonies. Furthermore, macrophage-associated Ia antigen was more efficient than B cell-associated Ia for these responses. The requirement for allogeneic Ia antigen was not absolute, since the colonies could easily be expanded when the cultures were supplemented with irradiated syngeneic PEC and the T cell mitogens, Con A or PHA. The effect of the mitogen was mediated via the T cells in the irradiated PEC, since removal of the T cells from these PEC markedly reduced the responses. Thus, it is likely that a nonspecific signal(s) presumably from T cells can promote proliferation of alloreactive T cell colonies in the absence of allogeneic Ia antigen. These results suggest 2 mechanisms of activation of these alloreactive T cells.     

Mutagenicity of 2-amino-N6-hydroxyadenine to TK6 human lymphoblast cells

TK6 human lymphoblast cells (tk +/-; hprt+) were treated with various concentrations of 2-amino-N6-hydroxyadenine (AHA) for 24 h. AHA was quite toxic to TK6 cells in the dose range 0-0.05 micrograms/ml, but additional toxicity was not observed between 0.05 and 0.10 micrograms/ml. AHA induced mutations at 2 distinct genetic loci: the autosomal thymidine kinase (tk) and the X-linked hypoxanthine-guanine phosphoribosyl transferase (hprt). Significant levels of both tk-NG mutants (normal growth rate of 16-18 h, colonies visible after 10-11 days incubation) and tk-SG mutants (slow growth rate of greater than 24 h, colonies visible after 18 days incubation) were induced. 15 hprt- mutants were isolated and analyzed by Southern blot. 8 of these had normal restriction fragment patterns after digestion with PstI, EcoRI, and HindIII, and were defined as 'point' mutations; the remaining 7 had partial deletions of the hprt gene. 32 tk- mutants were also isolated. 3 of 22 normal growth mutants and 6 of 10 slow growth mutants had lost the active tk allele. These data suggest that both point mutations and larger-scale alterations are induced by AHA.     

Intraclonal polymorphism in the bacterium Streptomyces ambofaciens ATCC23877: evidence for a high degree of heterogeneity of the wild type clones

In Streptomyces ambofaciens a genetic instability generates a high degree of polymorphism consisting of four main phenotypes: pigmented colonies (Pig(+) qualified as WT phenotype), pigment-defective colonies, pigmented colonies with pigment-defective sector and pigmented colonies with pigment-defective papillae. Molecular analysis of Pig(col)(-) and Pig(sec)(-) (pigment-defective mutant derived from a colony and a sector, respectively) produced by genetic instability and isolated in five Pig(+) subclones progenies revealed a new aspect of polymorphism in S. ambofaciens ATCC23877. Frequencies of Pig(col)(-) and Pig(sec)(-) mutants deleted at the chromosome ends varied from one WT progeny to another. Two main types of deleted mutants were observed: deleted for one or both chromosomal extremities. The relative proportion of these two categories differed according to the WT progeny. These results argue for heterogeneity of the WT clones, i.e., Pig(+) colonies, originated from S. ambofaciens ATCC23877.     

Attachment of the Yeast Rhodosporidium toruloides Is Mediated by Adhesives Localized at Sites of Bud Cell Development

The basidiomycetous yeast Rhodosporidium toruloides (anamorph, Rhodotorula glutinis) is a common phylloplane epiphyte with biocontrol potential. To understand how R. toruloides adheres to plant surfaces, we obtained nonadherent fungal mutants after chemical mutagenesis with methane-sulfonic acid ethyl ester. Sixteen attachment-minus (Att⁻) mutants were identified by three methods: (i) screening capsule-minus colonies for loss of adhesive ability; (ii) enrichment for mutants unable to attach to polystyrene; and (iii) selection for reduced fluorescence of fluorescein isothiocyanate-concanavalin A (Con A)-stained cells by fluorescence-activated cell sorting. None of the 16 mutants attached to polystyrene or barley leaves. The lectin Con A eliminated adhesion in all of the wild-type isolates tested. Hapten competition assays indicated that Con A bound to mannose residues on the cell surface. Adhesion of wild-type R. toruloides was transient; nonadhesive cells subsequently became adhesive, with bud development. All Att⁻ mutants and nonattaching wild-type cells lacked polar regions that stained intensely with fluorescein isothiocyanate-Con A and India ink. Lectin, enzyme, and chemical treatments showed that the polar regions consisted of alkali-soluble materials, including mannose residues. Tunicamycin treatment reduced wild-type adhesion, indicating that the mannose residues could be associated with glycoproteins. We concluded that compounds, including mannose residues, that are localized at sites of bud development mediate adhesion of R. toruloides to both polystyrene and barley leaf surfaces.     

Random mutagenesis of human cytochrome p450 2A6 and screening with indole oxidation products.

Cytochrome P450 (P450) 2A6 mutants from randomized libraries generated in the substrate recognition sequence (SRS) regions were screened in Escherichia coli on the basis of indole metabolism. SRS 3 and 4 libraries yielded colonies that produced indigo at least as well as wild-type (WT) P450 2A6, and some colonies were consistently more blue upon replating. One mutant, F209T, showed indole 3-hydroxylation WT. The double mutant L240C/N297Q consistently produced very blue colonies. Five mutants yielded mixtures of pigments from indole different than WT, as judged by visible spectra and HPLC of products. When bacteria expressing the mutants were grown in the presence of each of 26 substituted indoles, a variety of patterns of formation of different dyes was seen with several of the mutants. This approach has potential value in understanding P450 2A6 function and generating new dyestuffs and other products.     

Role of fadR and atoC(Con) mutations in poly(3-hydroxybutyrate-co-3-hydroxyvalerate) synthesis in recombinant pha+ Escherichia coli.

Recombinant Escherichia coli fadR atoC(Con) mutants containing the polyhydroxyalkanoate (PHA) biosynthesis genes from Alcaligenes eutrophus are able to incorporate significant levels of 3-hydroxyvalerate (3HV) into the copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)]. We have used E. coli fadR (FadR is a negative regulator of fatty acid oxidation) and E. coli atoC(Con) (AtoC is a positive regulator of fatty acid uptake) mutants to demonstrate that either one of these mutations alone can facilitate copolymer synthesis but that 3HV levels in single mutant strains are much lower than in the fadR atoC(Con) strain. E. coli atoC(Con) mutants were used alone and in conjunction with atoA and atoD mutants to determine that the function of the atoC(Con) mutation is to increase the uptake of propionate and that this uptake is mediated, at least in part, by atoD+. Similarly, E. coli fadR mutants were used alone and in conjunction with fadA, fadB, and fadL mutants to show that the effect of the fadR mutation is dependent on fadB+ and fadA+ gene products. Strains that were mutant in the fadB or fadA locus were unable to complement a PHA biosynthesis pathway that was mutant at the phaA locus (thiolase), but a strain containing a fadR mutation and which was fadA+ fadB+ was able to complement the phaA mutation and incorporated 3HV into P(3HB-co-3HV) to a level of 29 mol%.     

Breeding of wastewater treatment yeasts that accumulate high concentrations of phosphorus

Inorganic phosphate is an essential nutrient. In general, microorganisms take up phosphorus when the extracellular phosphorus concentration is low, but not when it is high. In Saccharomyces cerevisiae, the major phosphate transporters, such as Pho84p, and acid phosphatases (APases), such as Pho5p, are regulated in parallel by the phosphate signal transduction pathway (PHO pathway). We found that PHO mutants expressing PHO84 and PHO5, even under high-P conditions, could take up phosphorus at twice the rate of the wild-type strain. The regulatory pathway for phosphorus accumulation in two wastewater treatment yeasts, Hansenula fabianii J640 and Hansenula anomala J224-1, was found to be similar to that in S. cerevisiae. We screened for mutants of these yeasts that constitutively expressed APase. Such mutants formed blue colonies on high phosphorus concentration agar plates containing 5-bromo-4-chloro-3-indolylphosphate (X-phosphate). We found four mutants of H. fabianii J640 and one mutant of H. anomala J224-1 that accumulated from 2.2 to 3.5 times more phosphorus than the parent strains. The growth rates and abilities to remove dissolved total nitrogen and dissolved organic carbon of the mutants were similar to those of the parent strains. In addition, the mutants removed 95% of dissolved total phosphorus from shochu wastewater, while the parent strain removed only 50%.     

Tests of a cellular model for constant branch distribution in the filamentous fungus Neurospora crassa

The growth of mycelial fungi is characterized by the highly polarized extension of hyphal tips and the formation of subapical branches, which themselves extend as new tips. In Neurospora crassa, tip growth and branching are crucial elements for this saprophyte in the colonization and utilization of organic substrates. Much research has focused on the mechanism of tip extension, but a cellular model that fully explains the known phenomenology of branching by N. crassa has not been proposed. We described and tested a model in which the formation of a lateral branch in N. crassa was determined by the accumulation of tip-growth vesicles caused by the excess of the rate of supply over the rate of deposition at the apex. If both rates are proportional to metabolic rate, then the model explains the known lack of dependence of branch interval on growth rate. We tested the model by manipulating the tip extension rate, first by shifting temperature in both the wild type and hyperbranching (colonial) mutants and also by observing the behavior of both tipless colonies and colonyless tips. We found that temperature shifts in either direction result in temporary changes in branching. We found that colonyless tips also pass through a temporary transition phase of branching. The tipless colonies produced a cluster of new tips near the point of damage. We also found that branching in colonial mutants is dependent on growth rate. The results of these tests are consistent with a model of branching in which branch initiation is controlled by the dynamics of tip growth while being independent of the actual rate of this growth.     

Tests of a Cellular Model for Constant Branch Distribution in the Filamentous Fungus Neurospora crassa

The growth of mycelial fungi is characterized by the highly polarized extension of hyphal tips and the formation of subapical branches, which themselves extend as new tips. In Neurospora crassa, tip growth and branching are crucial elements for this saprophyte in the colonization and utilization of organic substrates. Much research has focused on the mechanism of tip extension, but a cellular model that fully explains the known phenomenology of branching by N. crassa has not been proposed. We described and tested a model in which the formation of a lateral branch in N. crassa was determined by the accumulation of tip-growth vesicles caused by the excess of the rate of supply over the rate of deposition at the apex. If both rates are proportional to metabolic rate, then the model explains the known lack of dependence of branch interval on growth rate. We tested the model by manipulating the tip extension rate, first by shifting temperature in both the wild type and hyperbranching (colonial) mutants and also by observing the behavior of both tipless colonies and colonyless tips. We found that temperature shifts in either direction result in temporary changes in branching. We found that colonyless tips also pass through a temporary transition phase of branching. The tipless colonies produced a cluster of new tips near the point of damage. We also found that branching in colonial mutants is dependent on growth rate. The results of these tests are consistent with a model of branching in which branch initiation is controlled by the dynamics of tip growth while being independent of the actual rate of this growth.