Homolog pairing and meiotic progression in Coprinus cinereus

We have used fluorescence in situ hybridization to examine homolog pairing during the synchronous meiosis of the basidiomycete Coprinus cinereus. Using spread preparations of meiotic nuclei, we confirmed previous studies that showed that at 6 h post-karyogamy essentially all meiotic nuclei are in pachytene. We found that homolog pairing occurs rapidly after karyogamy, that a 1 Mb chromosome does not associate more quickly than a 2.5 Mb chromosome, and that interstitial, single-copy sites can associate stably prior to nucleolar fusion. Analysis of two probes for the same pair of homologs revealed that by 4 h after karyogamy each chromosome examined was at least partially paired in all meiotic cells. In addition, these studies showed that chromatin condensation increases after pairing and that chromatin shows stable compaction at pachytene. Received: 4 January 1999; in revised form: 22 June 1999 / Accepted: 20 July 1999     

Silver staining of meiotic chromosomes in the fungus,Coprinus cinereus

We have taken advantage of the synchronous meiotic process in the basidiomycete Coprinus cinereus to develop a simple and rapid method to selectively stain meiotic chromosomes and nucleoli in this fungus without prior removal of the cell wall. Electron microscopic examination of these silver-stained chromosomes indicated that the lateral elements of the synaptonemal complexes were prominently stained, and terminal attachment plaques were apparent. We found that a translocation quadrivalent could be recognized easily in the light microscope using these methods. The procedures appear suitable for the characterization of chromosome rearrangements in this small genome, and should facilitate cytogenetic analysis in this fungus.     

Expression of flap endonuclease-1 during meiosis in a basidiomycete, Coprinus cinereus

In the basidiomycete Coprinus cinereus (C. cinereus), which shows a highly synchronous meiotic cell cycle, the meiotic prophase I cells demonstrate flap endonuclease-1 activity. To investigate its role during meiosis, we isolated a C. cinereus cDNA homolog of flap endonuclease-1 (CcFEN-1), 1377bp in length with the open reading frame (ORF) encoding a predicted molecular mass of 51 kDa. At amino-acid residues Glu276-Pro345, a specific inserted sequence composed of 70 amino acids rich in polar forms was found to exist, without sequence identity to other eukaryotic FEN-1 or the polar amino acid rich sequences found in C. cinereus PCNA and C. cinereus DNA ligase IV, although the lengths and percentages of polar amino acids were similar. Northern hybridization analysis indicated CcFEN-1 to be expressed not only in the pre-meiotic S phase but also in meiotic prophase I. The roles of CcFEN-1 during meiosis are discussed.     

Effect of light/dark cycles on wastewater treatments by microalgae

Chlorella kessleri was cultivated in artificial wastewater using diurnal illumination of 12 h light/12 h dark (L/D) cycles. The inoculum density was 10⁵ cells/mL and the irradiance in light cycle was 45 μmol m² s⁻¹ at the culture surface. As a control culture, another set of flasks was cultivated under continuous illumination. Regardless of the illumination scheme, the total organic carbon (TOC) and chemical oxygen demand (COD) was reduced below 20% of the initial concentration within a day. However, cell concentration under the L/D lighting scheme was lower than that under the continuous illuminating scheme. Thus the specific removal rate of organic carbon under L/D cycles was higher than that under continuous illumination. This result suggested thatC. kessleri grew chemoorganotrophically in the dark periods. After 3 days, nitrate was reduced to 136.5 and 154.1 mg NO₃ ⁻-N/L from 168.1 mg NO₃ ⁻-N/L under continuous illumination and under diurnal cycles, respectively. These results indicate nitrate removal efficiency under continuous light was better than that under diurnal cycles. High-density algal cultures using optimized photobioreactors with diurnal cycles will save energy and improve organic carbon sources removal.     

Inhibition of Coprinus cinereus by 5-fluoroindole

5-Fluoroindole (5FI) was more inhibitory than 5-fluorotryptophan (5FT) to Coprinus cinereus. A mutant blocked in the conversion of indole to tryptophan, but not one blocked earlier in the tryptophan pathway, was resistant to 5FI. This is consistent with the hypothesis that 5FI was converted in vivo to 5FT which inhibited growth.     

Efficiency of light utilization of Chlamydomonas reinhardtii under medium-duration light/dark cycles

The light regime inside a photobioreactor is characterized by a light gradient with full (sun)light at the light-exposed surface and darkness in the interior of the bioreactor. Consequently, depending on the mixing characteristics, algae will be exposed to certain light/dark cycles. In this study the green alga Chlamydomonas reinhardtii was cultivated under five different light regimes: (1) continuous illumination; (2) a square-wave light/dark cycle with a light fraction (epsilon) of 0.5 and a duration (t(c)) of 6.1 s; (3) epsilon=0.5, t(c)=14.5 s; (4) epsilon=0.5, t(c)=24.3 s and (5) epsilon=0.8, t(c)=15.2 s. The biomass yield on light energy, protein per photons, decreased under light/dark cycles (epsilon=0. 5) in comparison to continuous light (CL), from 0.207 (CL) to 0.117-0.153 g mol(-1) (epsilon=0.5). Concomitantly, the maximal specific photosynthetic activity, oxygen production per protein, decreased from 0.94 (CL) to 0.64-0.66 g g(-1) h(-1) (epsilon=0.5). Also the quantum yield of photochemistry, yield of the conversion of light energy into chemical energy, decreased from 0.47 (CL) to 0. 23 (epsilon=0.5, t(c)=24.3 s). Apparently, C. reinhardtii is not able to maintain a high photosynthetic capacity under medium-duration light/dark cycles and since specific light absorption did not change, light utilization efficiency decreased in comparison to continuous illumination.     

Efficient isolation and mapping of rad genes of the fungus Coprinus cinereus using chromosome-specific libraries.

We have constructed cosmid libraries from electrophoretically separated chromosomes of the basidiomycete Coprinus cinereus. These libraries greatly facilitate the isolation of genes by complementation of mutant phenotypes and are particularly useful for map-based cloning strategies. From a library constructed from two co-migrating C.cinereus chromosomes, we isolated a clone that complements the C.cinereus rad9-1 mutation. Examination of this clone showed that it complements both the repair and meiotic defects of this mutant. Restriction fragment length polymorphism mapping using a portion of this clone showed that it maps to the rad9 locus. In addition, a single copy of transforming DNA is sufficient to complement the rad9-1 defects. Thus, we believe we have cloned the rad9 gene itself. We also used a chromosome-specific library and backcrossed isolates to rapidly identify a cosmid clone which is tightly linked to the rad11 locus and is therefore a suitable starting point for a chromosome walk. These rapid methods of gene mapping and isolation should be applicable to any organism with separable chromosomes.     

The A mating type and blue light regulate all known differentiation processes in the basidiomycete Coprinus cinereus

Monokaryons of Coprinus cinereus constitutively form small spores (oidia) in the aerial mycelium. Some strains also produce large, inflated single cells (chlamydospores) at the agar/air interface, and hyphal aggregates (hyphal knots) that can develop into sclerotia. Monokaryons show various reactions upon transformation with heterologous A mating type genes. Production of oidia in such A-activated transformants is repressed in the dark and induced by blue light. Five of six monokaryons tested following transformation with A genes showed induced production of hyphal knots and sclerotia in the dark, and at least three strains showed enhanced chlamydospore production in the dark. Continuous incubation under blue light inhibited formation of hyphal knots, sclerotia and chlamydospores in both competent monokaryons and in A-activated transformants. On artificial medium and on a 12?h light/12?h dark regime, A-activated transformants of one distinct monokaryon (218) formed fruit-body primordia that were arrested in development before karyogamy. Our studies show that A mating type genes control all major differentiation processes in Coprinus, but whether developmental processes can proceed depends on the genetic background of the strain.     

A simple model of the growth of phytoplankton populations in light/dark cycles

Phytoplankton populations have been shown to be entrained byalternating periods of light and darkness in natural watersas well as in laboratory cultures. A simple model for the growthof such populations, as reflected by cell division, is presentedhere. The model takes as its structural unit the single cell,using Spudich and Sager's transition point hypothesis for thecoupling between received light and cell cycle progression.A stochastic component is also included to account for cell-to-cellvariability. The model predicts that the characteristics ofcell division patterns in populations entrained by photocyclesdepends mainly on the position of the transition point withinthe cell cycle, rather than on the characteristics of the photocyclicregime. The model simulates successfully the major featuresof observed division patterns of several phytoplankton species.In addition, the model can be used to predict division patternsin high frequency photocycles and during transients inducedby shifts in light regime. Under these conditions, the simulatedpatterns are also consistent with the hypothesis of a circadianclock controlled cell cycle, except in the case of free runningtransients. ¹Present address: Station Biologique Roscoff, CNRS, Roscoff29211, France     

Entrainment of 2 subjective nights by daily light:dark:light:dark cycles in 3 rodent species

Recent work with exotic 24-h light:dark:light:dark (LDLD) cycles indicates surprising flexibility in the entrainment patterns of Syrian hamsters. Following exposure to an LDLD cycle, hamsters may adopt a form of rhythm splitting in which markers of subjective night (e.g., activity, melatonin) are expressed in each of the twice daily scotophases. This pattern contrasts markedly with that of conventionally entrained hamsters in which markers of subjective night are expressed once daily in only 1 of the 2 dark periods. The "split" entrainment pattern was examined further here in Syrian and Siberian hamsters and in mice exposed to LDLD 7:5:7:5, a condition that reliably induces split activity rhythms in all 3 species. The phase angle of entrainment and activity duration were generally similar comparing the 2 daily activity bouts in each species. The stability of this split entrainment state was assessed by deletions of photophases on individual days, by exposure to skeleton photoperiods, and by transfer to constant darkness. As in Syrian hamsters, the one-time substitution of darkness for one 7-h photophase did not grossly alter activity patterns of Siberian hamsters but acutely disrupted the split rhythms of mice. Skeleton light pulses of progressively shorter duration did not significantly alter split entrainment patterns of either Syrian or Siberian hamsters. Both species continued to exhibit stable entrainment with activity expressed in alternate scotophases of an LD 1:5 cycle presented 4 times daily. In contrast, the split activity rhythms of mice were not maintained under skeleton pulses. In constant darkness, rhythms of Siberian hamsters remained distinctly split for a minimum of 2 cycles. Split entrainment to these novel LDLD and 4-pulse skeleton lighting regimes demonstrates a marked degree of plasticity common to the circadian systems of several rodent species and identifies novel entrainment patterns that may be reliably elicited with simple environmental manipulations. Inter- and intraspecific differences in the stability of split activity rhythms likely reflect differences in coupling interactions between the component circadian oscillators, which, adopting separate phase relations to these novel LD cycles, yield a split entrainment pattern.     

DNA-mediated transformation of the basidiomycete Coprinus cinereus.

We have developed a simple and efficient transformation system for the agaric fungus, Coprinus cinereus. Protoplasts were prepared from asexual spores that harbor one or two mutations in the structural gene for tryptophan synthetase. The protoplasts can be stably transformed using the cloned Coprinus gene at a frequency of 1 in 10(4) viable protoplasts. A variety of molecular events accompanies the formation of stable transformants, including insertion of the transforming DNA at the homologous locus. The transforming DNA is stable through cell division, mating, fruiting body formation, and meiosis.     

Scale-up aspects of photobioreactors: effects of mixing-induced light/dark cycles

The green micro-algae Chlamydomonas reinhardtiiand Dunaliella tertiolecta were cultivated undermedium-duration square-wave light/dark cycles with acycle time of 15 s. These cycles were used to simulatethe light regime experienced by micro-algae inexternally-illuminated (sunlight) air-lift loopbioreactors with internal draft tube. Biomass yieldin relation to light energy was determined as gprotein per mol of photons (400–700 nm). Between 600and 1200 mol m^-2 s^-1 the yield at a10/5 s light/dark cycle was equal to the yield atcontinuous illumination. Consequently, provided thatthe liquid circulation time is 15 s, a considerabledark zone seems to be allowed in the interior ofair-lift loop photobioreactors (33% v/v) without lossof light utilization efficiency. However, at a 5/10 slight/dark cycle, corresponding to a 67% v/v darkzone, biomass yield decreased. Furthermore, bothalgae, C. reinhardtii and D. tertiolecta,responded similarly to these cycles with respect tobiomass yield. This was interesting because they werereported to exhibit a different photoacclimationstrategy. Finally, it was demonstrated that D.tertiolecta was much more efficient at low (average)photon flux densities (57–370 mol m^-2s^-1) than at high PFDs (> 600 mol m^-2s^-1) and it was shown that D. tertiolectawas cultivated at a sub-optimal temperature (20 ^°C).     

Proliferating cell nuclear antigen from a basidiomycete, Coprinus cinereus. Alternative truncation and expression in meiosis.

The primary purpose of the present study was to investigate whether DNA replication at meiotic prophase also requires replication factors, especially proliferating cell nuclear antigen (PCNA). We cloned PCNA cDNAs (CoPCNA) from a cDNA library made from basidia of the basidiomycete, Coprinus cinereus. Interestingly, although CoPCNA is a single-copy gene in the genome, two different PCNA cDNA species were isolated using degenerate primers and a meiotic cDNA library, and were designated as CoPCNA-alpha and CoPCNA-beta. CoPCNA-beta was made by truncating at specific sites in CoPCNA-alpha mRNA, 5'-AAGAAGGAGAAG-3' and 5'-GAAGAGGAAGAA-3'. Both of these sequences were present in exon IV in the genomic sequence, and interestingly the former was the same as the inverse sequence of the latter. CoPCNA-alpha was 107 amino acids larger than human PCNA, and so the 107 amino-acid sequence was inserted in a loop, the so-called D2E2 loop, in human PCNA. Northern blotting analysis indicated that CoPCNA was expressed not only at premeiotic S but also at the meiotic prophase stages such as leptotene and early zygotene, just before and when karyogamy occurs and the homologous chromosomes pair. Western blotting analysis using anti-(CoPCNA-alpha) Ig revealed that at least two CoPCNA mRNAs before and after truncation were translated at the meiotic prophase as CoPCNA-alpha and CoPCNA-beta.     

Inheritance of DNA methylation in Coprinus cinereus.

We examined the inheritance of 5-methylcytosine residues at a centromere-linked locus in the basidiomycete Coprinus cinereus. Although methylated and unmethylated tracts were inherited both mitotically and meiotically the lengths of these tracts were variable. This variation was not confined to any one phase of the life cycle of the organism, and it usually involved the simultaneous de novo methylation of at least four HpaII-MspI sites. We also found that the higher levels of methylation at this locus were transmitted through meiosis, regardless of the level of methylation of the homologous chromosome.     

The rad3-1 mutant is defective in axial core assembly and homologous chromosome pairing during meiosis in the basidiomycete Coprinus cinereus

We have utilized spreading methods as well as serial sectioning three-dimensional reconstruction to examine meiotic chromosome behavior in cells homozygous for the rad3-1 mutation in Coprinus cinereus. Comparison of 42 wild-type nuclei that had been spread, stained with silver, and viewed by electron microscopy with 30 mutant nuclei treated in the same manner revealed several defects in the mutant. Axial core formation was defective in the mutant, although limited side-by-side association of axial cores was observed. To detect any differences in three-dimensional architecture between the wild-type and mutant nuclei, we reconstructed three of the former and six of the latter after serial sectioning. It was not possible to trace the expected number of axial cores from section to section in the mutant, although some tripartite synaptonemal complex was observed. Many axial core ends failed to terminate in the nuclear envelope in the mutant. This spectrum of defects (incomplete axial core assembly with some tripartite synaptonemal complex formation) had not been observed previously in either C. cinereus or other systems. We conclude that this combination of spreading and sectioning methods is very useful for analysis of meiotic mutants. © 1993 Wiley-Liss, Inc.