Genomic organization of the fungus Phycomyces

The fungus Phycomyces blakesleeanus has a relatively small genome, 30 megabases (Mb), with a low guanine and cytosine (G+C) content, 35%; the coding sequences cloned to date all have a G+C content of about 50%. In order to investigate the organization of the genome of this fungus, we have cloned and sequenced 251 DNA fragments. One hundred and twenty-six clones were obtained by digestion with MspI (target sequence 5′-CCGG-3′) and 125 random clones were obtained by sonication. The average length of sequence obtained was about 200 base pairs (bp) and the total length was about 50 kilobases (kb). The G + C content is not homogeneous throughout the genome: sequences obtained after digestion with MspI have an average of 5% more G + C content than the random fragments, and are enriched in coding sequences. Fourteen MspI fragments show similarities to known proteins and 21 encode ribosomal RNA (rRNA). By contrast, only three of the random fragments are similar to known proteins and only one to a rRNA. We conclude that the Phycomyces genome is composed of G+C-rich genes surrounded by G+C-poor areas. Two clones have similarities to the transposase of the transposon Tcl from Caenorhabditis elegans. This result suggests the presence of a high copy number of a Tcl-like transposable element in the Phycomyces genome. Another clone was similar to the transposon Txl from Xenopus laevis. A novel repetitive nt sequence has been characterized; about 5% of the total genome is a repetition of any of two consensus sequences of 31 by named PrAI and PrA2.     

A cytoplasmically inherited mutation in the fungus Phycomyces blakesleeanus

Fourteen mutants of the fungus Phycomyces blakesleeanus, showing high levels of resistance to copper, were isolated. In all the mutants, copper resistance behaved as a very variable and unstable trait. In the mutant strain MU102, the mutation was demonstrated to be cytoplasmically inherited. In addition, this mutant strain differed from the wild-type in growth, respiration rate, and shape and viability of spores.     

Expression of Tn5-derived kanamycin resistance in the fungus Phycomyces blakesleeanus

Summary A plasmid, carrying the Tn5 gene for kanamycin resistance lacking its own promoter, has successfully been used in the selection of DNA sequences of the fungus Phycomyces blakesleeanus having promoter activity in Escherichia coli. Many of these sequences were also effective in promoting resistance to kanamycin when the corresponding chimeric plasmids were introduced in the fungus via spheroplast transformation. The selected phenotype was easily propagated through vegetative spores and behaved as a stable character since it was not appreciably lost in the absence of selection.     

Mossbauer studies of anhydrous hemoglobin and its subunits

The similarities in the Mossbauer spectra of Hb and its α and β subunits, both in the oxygenated and deoxygenated states, were observed. The relative intensities of the absorption dips in the Mossbauer spectra of ⁵⁷Fe in anhydrohemoglobin (AHb) and anhydrous separated alpha (Aα) and beta (Aβ) chain samples were found to be very sensitive to the conditions of samples preparation. This indicates the formation of hemochromogen as an impurity in the absorbers during the process of preparation. When contributions due to the impurity in the samples were subtracted, Mossbauer spectra of ⁵⁷Fe in AHb, Aα, and Aβ remained similar. Therefore, the electronic structure of the iron cation is the same for Hb and its subunits in the anhydrous state also.     

Lactate dehydrogenase in Phycomyces blakesleeanus.

1. An NAD-specific L(+)-lactate dehydrogenase (EC 1.1.1.27) from the mycelium of Phycomyces blakesleeanus N.R.R.L. 1555 (-) was purified approximately 700-fold. The enzyme has a molecular weight of 135,000-140,000. The purified enzyme gave a single, catalytically active, protein band after polyacrylamide-gel electrophoresis. It shows optimum activity between pH 6.7 and 7.5. 2. The Phycomyces blakesleeanus lactate dehydrogenase exhibits homotropic interactions with its substrate, pyruvate, and its coenzyme, NADH, at pH 7.5, indicating the existence of multiple binding sites in the enzyme for these ligands. 3. At pH 6.0, the enzyme shows high substrate inhibition by pyruvate. 3-hydroxypyruvate and 2-oxovalerate exhibit an analogous effect, whereas glyoxylate does not, when tested as substrates at the same pH. 4. At pH 7.5, ATP, which inhibits the enzyme, acts competitively with NADH and pyruvate, whereas at pH 6.0 and low concentrations of ATP it behaves in a allosteric manner as inhibitor with respect to NADH, GTP, however, has no effect under the same experimental conditions. 5. Partially purified enzyme from sporangiophores behaves in entirely similar kinetic manner as the one exhibited by the enzyme from mycelium.     

A gene for carotene cleavage required for pheromone biosynthesis and carotene regulation in the fungus Phycomyces blakesleeanus

Mating and sexual development in fungi are controlled by molecular mechanisms that are specific for each fungal group. Mating in Phycomyces blakesleeanus and other Mucorales requires pheromones derived from β-carotene. Phycomyces mutants in gene carS accumulate large amounts of β-carotene but do not enter the sexual process. We show that carS encodes a β-carotene-cleaving oxygenase that catalyzes the first step in the biosynthesis of a variety of apocarotenoids, including those that act as pheromones. Therefore carS mutants cannot stimulate their sexual partners, although they respond to them. CarS catalyzes the biosynthesis of a β-ring-containing apocarotenoid that inhibits the activity of the carotenogenic enzyme complex in vegetative cells and provides a feedback regulation for the β-carotene pathway. The carS gene product is a keystone in carotenogenesis and in sexual reproduction.     

Carotene-superproducing strains of Phycomyces.

Production of beta-carotene by wild-type Phycomyces blakesleeanus can be stimulated by light, chemicals, regulatory mutations, and sexual interaction between mycelia of opposite sex. Through genetic manipulations, we have isolated strains which have simultaneously and constitutively incorporated several of these stimulatory effects. In the dark and in a simple medium, some of the strains produce up to 25 mg of beta-carotene per g (dry weight), or about 500 times the wild-type production under the same conditions. High lycopene-producing strains have also been isolated by using carR mutants, which are blocked in the conversion of lycopene to beta-carotene. These strains should be useful in both industrial production of these pigments and basic research related to carotenogenesis.     

Olfactory responses of Phycomyces blakesleeanus.

Upon exposure to low levels of various volatile compounds such as n-heptanol, methanol, CHCl₃, mercaptoheptane, etc., the sporangiophore of $\text{Phycomyces}\text{blakesleeanus}$ responds with a transient and reproducible decrease in its elongation rate. All 22 volatile substances tested (except H₂O) elicited negative responses. The amplitude of the responses depends on the compound and its concentration. A characteristic concentration, required for 50% inhibition, correlates remarkable well with the human olfactory threshold (coefficient of correlation r = 0.89 (P < 0.001)). Perhaps some process in olfaction is common to this fungus and higher systems.     

Avoidance of Phycomyces in a controlled environment.

The sporangiophore of the fungus Phycomyces bends away from nearby objects without ever touching them. It has been thought that these objects act as aerodynamic obstacles that damp random winds, thereby generating asymmetric distributions of a growth-promoting gas emitted by the growth zone. In the interest of testing this hypothesis, we studied avoidance in an environmental chamber in which convection was suppressed by a shallow thermal gradient. We also controlled pressure, temperature, and relative humidity of the air, electrostatic charge, and ambient light. A protocol was established that yielded avoidance rates constant from sporangiophore to sporangiophore to within +/- 10%. We found that avoidance occurred at normal rates in the complete absence of random winds. The rates were smaller at 100% than at lower values of relative humidity, but not by much. Remarkably, at a distance as great as 0.5 mm, avoidance from a 30-micron diam glass fiber (aligned parallel to the sporangiophore) was about the same as that from a planar glass sheet. However, the rate for the fiber fell more rapidly with distance. The rate for the sheet remained nearly constant out to approximately 4 mm. We conclude that avoidance depends either on adsorption by the barrier of a growth-inhibiting substance or emission by the barrier of a growth-promoting substance; it cannot occur by passive reflection. Models that can explain these effects are analyzed in the Appendix.