Genetic analysis of the yeastCandida maltosa by means of induced parasexual processes

The usefulness of hybridization by protoplast fusion and mitotic segregation for the genetic analysis of the imperfect fodder yeastCandida maltosa was tested. Mitotically stable fusion hybrids were obtained with frequencies between 10^–6 and 10^–7. Complementation tests were performed by protoplast fusion. Substances that are known to induce frequent mitotic segregation in other yeast species such as benomyl, p-fluorophenylalanine, and acriflavine were ineffective inC. maltosa. UV irradiation induced mitotic segregation in up to 10%. This agent induced mainly mitotic crossing over inC. maltosa. Our data enabled the construction of the linkage group I with the sequenceCEN-ade-26-pro-1.     

Genetic analysis of Aspergillus niger: Isolation of chlorate resistance mutants, their use in mitotic mapping and evidence for an eighth linkage group

Summary This paper describes the use of chlorate resistant mutants in genetic analysis of Aspergillus niger. The isolated mutants could be divided into three phenotypic classes on the basis of nitrogen utilization. These were designated nia, nir and cnx as for Aspergillus nidulans. All mutations were recessive to their wild-type allele in heterokaryons as well as in heterozygous diploids. The mutations belong to nine different complementation groups. In addition a complex overlapping complementation group was found. Evidence for the existence of eight linkage groups was obtained. Two linked chlorate resistance mutations and two tryptophan auxotrophic markers, which were unlinked to any of the known markers (Goosen et al. 1989), form linkage group VIII. We used the chlorate resistance mutations as genetic markers for the improvement of the mitotic linkage map of A. niger. We determined the linear order of three markers in linkage group VI as well as the position of the centromere by means of direct selection of homozygous cnxA1 recombinants. In heterozygous diploid cultures diploid chlorate resistant segregants appeared among conidiospores with a frequency of 3.9×10^–2 (cnxG13 in linkage group I) to 2.1 × 10^–2 (cnxD6 in linkage group 111). The mean frequency of haploid chlorate resistant segregants was 1.3 × 10^–3. The niaD1 and niaD2 mutations were also complemented by transformation with the A. niger niaD ⁺ gene cloned by Unkles et al. (1989). Mitotic stability of ten Nia⁺ transformants was determined. Two distinct stability classes were found, showing revertant frequencies of 5.0 × 10^–3 and 2.0 × 10^–5 respectively.     

Extrachromosomal inheritance inSchizosaccharomyces pombe

Summary Spontaneous chloramphenicol (cap ^r)- and erythromycin (ery ^r)-resistant mutants were isolated from strain ade7–50 h ^- and the antimycin-resistant mutant ana ^r-8 ade 7–50 h^- of Schizosaccharomyces pombe (Sch. p.). By mitotic segregation analysis all 154 cap ^r- and 120 ery ^r-mutants derived from ade 7–50 h ^- proved to be recessive chromosomal, whereas all 108 cap ^r- and 200 ery ^r-mutants originating from ana ^r-8 were extrachromosomally inherited. The rate of spontaneous cap ^r- and ery ^r-mutants was about hundredfold in ana ^r-8 compared to ade 7–50 h ^-. Growth of cap ^r-and ery ^r-mutants was not inhibited by chloramphenicol or erythromycin, respectively, in glucose-medium and only slightly in glycerol-medium at concentrations which completely inhibited ana ^r-8. By mitotic segregation-, tetrad-, and mitotic haploidization-analysis the extrachromosomal inheritance of mutants derived from ana ^r-8 was established. Segregational patterns of cap ^r- and ery ^r-determinats during mitosis, meiosis, and mitotic haplidization of diploids are discussed.     

Co-segregation of nitrate-reductase activity and normal regeneration ability in selfed sibs of Nicotiana plumbaginifolia somatic hybrids,heterozygotes for nitrate-reductase deficiency

Summary The nitrate-reductase (NR) defective cell lines of Nicotiana plumbaginifolia isolated in our laboratory could not be regenerated into plants on the standard medium (Márton et al. 1982 a). The normal regeneration potential, however, was restored in somatic hybrids obtained by fusing the NR^– (green) lines with a pigment deficient (P^–), but NR⁺ line, A28. Somatic hybrid plants were fertile in two combinations (A28 + NA9 and A28 + NX9). As expected, segregation for NR^– and P^– was found after selfing the somatic F1 (SF1) obtained by protoplast fusion, and in the F2. The variable segregation ratios are explained by chromosome abnormalities. Co-segregation of the NR^– phenotype and the altered response to shoot induction on standard medium suggest the involvement of the nitrate-assimilatory pathway in determining shoot regeneration ability.     

Recombinagenicity of caffeine for Candida albicans

Caffeine at concentrations of 0.5 × 10^–2 M or higher inhibited cell replication and induced gene segregations in Candida albicans cultured on defined complete medium. Both responses increased incrementally with increasing caffeine concentrations, and were more severe during incubation at 37 °C than 25 °C; at 37 °C, caffeine levels above 1.5 × 10^–2 M caused cellular inactivation. Caffeine effects occurred only under conditions permitting cell growth, and their magnitudes were greater for unbudded than budding cells, were influenced by cellular genetic backgrounds, and were unaffected by the presence of adenine in the medium. Evaluations of segregations for recessive auxotrophic markers of a four member linkage group carried heterozygously in a cis arrangement in treated cells established that induced segregants arise through either reciprocal or nonreciprocal recombinations. The frequency distributions of classes of reciprocal and nonreciprocal recombinants for these markers conformed with those previously obtained following induction by ultraviolet radiation, indicating that the probabilities of recombinational events within the chromosomal regions defined by the markers are not biased by the differences in kinds of initial DNA lesions caused by the two recombinagens. A panel of four protoplast fusion hybrids considered deficient for DNA repair because of enhanced susceptibilities to UV induced cellular inactivation and mitotic recombination exhibited corresponding increased sensitivities to caffeine, signifying that DNA damage induced by caffeine is subject to repair. Caffeine did not affect behavior of a variant strain exhibiting high frequency phenotypic switching between minute smooth and large rough colonial forms, and no evidence for mutagenicity of the drug was obtained with systems for detection of forward or reverse mutations. The mechanism of caffeine's recombinagenicity, and the implications of that property for genetic studies of C. albicans are discussed.     

Parallel plastic tank experiments with cultures of marine diatoms

The reproductibility of tank experiments concerning unicellular marine algal development was analyzed by means of parallel experiments with cultures ofThalassiosira rotula andSkeletonema costatum, using large flexible plastic tanks under semi-natural conditions. The tanks (3–4 m³, 4–5 m deep) were exposed in the German Bight at a station in the outer harbour of helgoland. The water was obtained from the open North Sea in towable tanks; it was filtered (plate filter), enriched with nitrate (20–30 gat dm^–3), phosphate (1.3–2.3 gat dm^–3) and silicate (15–23 gat dm^–3)-nearly natural springtime concentrations in this area-and inoculated with 10³–10⁵ cells dm^–3. The water was mixed with non-metal stirring equipment. Within 5 days, concentrations of 10⁶–10⁷ cells dm^–3 in an exponential growth phase were obtained. In experiments withT. rotula a parallel development was achieved in spite of some contamination by surrounding water. This is the case for nearly all parameters analyzed (nutrient salts, phytoplankton, bacteria, C, N and particulate carbohydrates). The heterotrophic bacteria, which were determined by means of the plate method, reached concentrations of up to 10⁶ (T. rotula) and 10⁵ (S. costatum) CFU cm^–3, respectively. They showed a consistent retrograde development at diatom concentrations above a certain level. The crop did not increase again until the diatoms had reached the stationary phase. During exponential growth ofT. rotula (G=8.9–11.7 h) a partially synchronous cell division was observed. There were also rhythms with respect to cell size (pervalvar axes) and chain length (number of cells). For the experiments withS. costatum (G=10–11.4 h) diurnal variations of cell size and chain length occurred. The present results indicate acceptable reproducibility of algal development and related phenomena in enclosed water bodies.     

Batch cultures of recombinant Lactococcus lactis subsp. lactis in a stirred fermentor

Summary The transfer of plasmids was studied in a stirred fermentor in the course of mixed batch cultures combining recombinant strains of Lactococcus lactis subsp. lactis (donor strains) with L. lactis subsp. lactis CNRZ 268M3 (recipient strain). Donor strains contained one or two of the following plasmids (coding for erythromycin or chloramphenicol resistance): pIL205 (self-transmissible), pIL252, pIL253 (non-transmissible but mobilizable by pIL205, respectively small and large copy number) and pE194 (inserted in the chromosome). Only self-transmissible plasmid pIL205 was transferred, with frequencies ranging from 10^–7 to 10^–8 after 12 h of fermentation. These frequencies were 60–400 times lower than in unstirred M17 broth and 100 000 times lower than on agar medium. In the latter case, non-transmissible plasmids pIL252 and pIL253 were mobilized by pIL205 with a frequency of about 10^–5–10^–6. Correspondence to: C.-Y. Boquien     

Meiofauna assemblages discharged by springs from a phreatic aquifer system in the Netherlands

Groundwater meiofauna washed out of springs was studied by means of spring water filtration. The principal interest was a quantitative analysis of the number and diversity of outwash fauna in relationship to hydrological conditions. In addition, a comparative analysis was made of outwash fauna of different outlets of the same hydrological system. The spring complex studied is part of the Centraal Plateau in the southern-most part of The Netherlands. This area is characterized by, for The Netherlands, relatively high hydraulic heads and large permeability of aquifers. Influence of human activities (agriculture) in the recharge area was demonstrated by elevated concentrations of nitrate, chloride, and sulphate in the spring waters. No traces of groundwater pollution by heavy metals or pesticides were found. Temporal variation of meiofauna outwash was studied in a single rheocrene spring during one year. This rheocrene had a relatively large and constant discharge (1300±8 ¦h^–1). Meiofauna numbers fluctuated between 110–240 ind. m^–3. Considerable differences in meiofauna numbers in a one-off analysis of different springs (rheocrenes and helocrenes) were found. Numbers ranged from 140 to 5800 org. m^–3 and an inverse relationship with the amount of water discharged was shown. Organisms in the water filtrates were of multiple origin: aquifer(e.g. Niphargus, Parastenocaris), benthic spring head habitats (e.g. Gammarus, Chironomidae), and (saturated) soils near the outlets (e.g. Criconomatidae). Meiofauna abundance and composition is discussed in relation to hydrology and spring head morphology. A semi-quantitative analysis of outwash aquifer fauna was masked by the presence of epigean elements or elements of unclear origin. The outwash fauna of rheocrenes of large discharge supposedly is the most representative for the aquifer. Adaptations of hypogean populations to oligotrophic porous environments and the consequences for drift of juvenile stages is discussed.     

Isolation of Aneuploid-Generating Mutants of ASPERGILLUS NIDULANS, One of Which Is Defective in Interphase of the Cell Cycle

A method is described for isolating mutants potentially defective in loci involved in mitotic chromosome segregation. Conditional lethal, heat-sensitive (42°) mutants were assayed at a subrestrictive temperature of 37° for an inflated production of colonies displaying phenotypes and behavior patterns of whole chromosome aneuploids. Of 14 mutants, three showed specificity for one disomic phenotype, whereas 11 generated colonies mosaic for different aneuploid phenotypes. This latter group is designated hfa ( high frequency of aneuploid). For ten of the 11 mutants temperature sensitivity and aneuploid production cosegregated, indicating a single mutation in each. These mutations were recessive and nonallelic. Analysis was concentrated on the hfaB3 mutation which is mapped to chromosome VI tightly linked to the methB and tsB loci. The disruptive influence of hfaB3 on mitosis at 37° was shown by (1) ploidy and whole chromosome-type segregation of markers in the breakdown sectors of phenotypically aneuploid colonies obtained from multiply marked homozygous hfaB3 disploids; (2) a high frequency of haploid and nondisjunctional diploid segregants among spontaneous yellow-spored parasexual recombinants taken from green-spored homozygous hfaB3 diploids. The mutation had no effect on meiotic chromosome segregation at 37°. The single interphase nucleus in germlings at 42°, coupled with changes in the mitotic index in temperature exchange experiments, showed hfaB3 to arrest the cell cycle in interphase at restrictive temperature. A conclusion drawn is that the hfaB gene product is required both for entry into mitosis and for normal chromosome segregation in dividing nuclei.     

Size selectivity of aqueous pores in astomatous cuticular membranes isolated from<Emphasis Type="Italic"> Populus canescens</Emphasis> (Aiton) Sm. leaves

Little is known about the permeability of plant cuticles to ionic molecules with hydration shells that render them lipid insoluble and limit their diffusion to narrow aqueous pores. Therefore, the permeation of cuticular membranes to ionised calcium salts with anhydrous molecular weights ranging from 111 to 755 g mol^–1 was studied. Penetration was a first-order process and rate constants (k) (proportional to permeability) decreased exponentially with molecular weight. Plots of log k vs. molecular weight had slopes of –2.11×10^–3 and –2.80×10^–3, respectively, depending on the year in which the cuticular membranes were isolated. This corresponds to decreases in permeability by factors of about 7 to 13 when molecular weight increased from 100 to 500 g mol^–1. This size selectivity is small compared to the dependence on molecular weight of solute mobility in Populus cuticles. A decrease in mobility of neutral molecules by more than 3 orders of magnitude has been reported [A. Buchholz et al. (1998) Planta 206:322–328] for the same range of molecular weights. Hence, discrimination of large ionic species diffusing in aqueous pores (polar pathway) is much smaller than that for neutral solutes diffusing in cutin and waxes (lipophilic pathway). This indicates that formulating large solutes as ionic species would be advantageous.Abbreviation CM Cuticular membrane     

Characterization of Parasarcophaga heterochromatin

The heterochromatin of neuroblast metaphase chromosomes in six sympatric species of Parasarcophaga was characterized by C-, N-, quinacrine (Q)-, Hoechst 33258 (H)-, Hoechst/actinomycin D (H/AMD)- and acridine orange (AO)-banding. The autosomes and the sex chromosomes in all the six species are characterized by the presence of C- and N-positive heterochromatin. The N-bands lie within the limits of the C⁺ heterochromatin. AT-specific fluorochromes further differentiate heterochromatin into Q⁺H⁺,Q^–H⁺ and Q^–H^– types and the repetitive nature of these regions is indicated by their fast reassociating nature with AO-staining. Thus, on the basis of these results, the heterochromatin of mitotic chromosomes in Parasarcophaga can be classified into C⁺N⁺Q⁺H⁺, C⁺N⁺Q^–H^–, c⁺n^–q^–h^– and C^–N^–Q^–H⁺ types.     

Extrachromosomal inheritance of nalidixic acid resistance in the petite negative yeast Schizosaccharomyces pombe

Summary Spontaneous mutants resistant to nalidixic acid (NAL) were isolated from the petite negative yeast Schizosaccharomyces pombe (S. pombe). One of these mutants, resistant to 200 g/ml NAL, nal ^r–Y13, was characterized both genetically and biochemically. The extrachromosomal inheritance of this mutation was demonstrated both by mitotic segregation and by mitotic haploidization analysis. In the wild-type, NAL at a concentration of 100 g/ml almost completely inhibits incorporation of [¹⁴C]adenine in total DNA as well as in mitochondrial DNA. In the NAL-resistant mutant both total DNA synthesis and mitochondrial DNA synthesis were resistant to the drug. These results are discussed in view of previously published findings on the close interaction between the two DNA synthesizing systems in S. pombe.     

Study of RecA-Independent Homologous Recombination and a Chromosomal Rearrangement in the Escherichia coli Strain Carrying an Extended Heterozygous Tandem Duplication

A heterozygous tandem duplication in the Escherichia coli deo operon region deoAdeoB::Tn5/ deoCdeoDthr::Tn9 with the total length approximately 150 kb, which was obtained in the conjugational mating in the HfrH strain, was examined. By means of digestion with the NotI enzyme, pulsed-field gel electrophoresis, and the conjugational transfer of the duplication in the F^– strain, the chromosomal rearrangement, which occurred in the duplication region upon its stabilization in the bacterial genome, was studied. In a more stable strain, two new NotI sites were shown to appear in the chromosomal region located close to the duplication, which might have resulted from the transposition of the IS50 sequence from Tn5. The data were also obtained indicating the possibility of secondary transposition of the chromosomal segment between the two new NotI sites (approximately 30 kb) in the region located near the duplication. With the use of rec ⁺ and recA strains, two types of haploid and diploid segregants generated by the duplication were studied: DeoD⁺ (the deoD⁺ allele is not expressed in the original duplication due to the polar effect of the deoB::Tn5 insertion) and DeoC DeoD. The segregation of DeoD⁺ clones was shown to be RecA-dependent, whereas the DeoC DeoD segregants selected on the medium that contained thymine at a low concentration (i.e., under conditions of thymine starvation) appeared at a rather high frequency. However, the relative frequency of haploid clones, which have lost the duplication, strongly decreased in the recA genome among segregants of both types.     

Feeding in Euchlanis dilatata lucksiana Hauer on filamentous cyanobacteria and a prochlorophyte

Ingestion and assimilation rates of Euchlanis dilatata lucksiana Hauer, isolated from Lake Loosdrecht (The Netherlands) and cultured on lake water (seston < 33 µm), were measured in the laboratory using the ¹⁴C-tracer technique. The five taxa used as tracer foods, together with 6–7 mg C l^–1 of lake seston in each case, included four species of filamentous cyanobacteria (Oscillatoria redekei, O. limnetica, Aphanizomenon flos-aquae, Anabaena PCC 7120) and a prochlorophyte (Prochlorothrix hollandica). Except Anabaena, they are all commonly encountered in eutrophic Loosdrecht lakes, including Lake Loosdrecht, and their dimensions ranged between 150 and 250 µm in length and 2 and 3.5 µm in width. The small and large Euchlanis used as experimental animals had mean lengths of 217–223 µm and 276–305 µm, respectively. Euchlanis fed on all the taxa offered as food. Clearance rates ranged from 51 to 99 µl ind^–1 d^–1 for the large animals and from 22 to 41 µl ind^–1 d^–1 for the small animals. The highest ingestion rate observed, 1.7 µg ind^–1 d^–1, was for the large animals feeding on Aphanizomenon. The daily ration for both size classes far exceeded 100% of body weight, reaching up to 690% for the small animals feeding on Aphanizomenon. The small animals generally appeared to assimilate the ingested food more efficiently (assimilation efficiencies: 37–100%) than the large animals (34–77%). Compared with an earlier study in which only lake seston (<33 µm) was used as food, the specific clearance rates of Euchlanis on the cyanobacteria and Prochlorothrix were generally somewhat lower.     

Factors regulating summer phytoplankton in a highly eutrophic Antarctic lake

Lakes from Maritime Antarctica are regarded as systems generally inhabited by metazoan plankton capable of imposing a top-down control on the phytoplankton during short periods, while lakes from Continental Antarctica lacking these communities would be typically controlled by scarcity of nutrients, following a bottom-up model. Otero Lake is a highly eutrophic small lake located on the NW of the Antarctic Peninsula, which has no metazoan plankton. During summer 1996, we studied the density, composition and vertical distribution of the phytoplankton community of this lake with respect to various abiotic variables, yet our results demonstrated neither light nor nutrient limitation of the phytoplankton biomass. Densities of heterotrophic nanoflagellates (HNAN) and ciliates from three different size categories were also studied. Extremely low densities of HNAN (0–155 ind. ml^–1) could be due to feeding competition by bacterivore nanociliates and/or predation by large ciliates. A summer bloom of the phytoflagellate Chlamydomonas aff. celerrima Pascher reached densities tenfold those of previous years (158.10³ ind. ml^–1), though apparently curtailed by a strong peak of large ciliates (107 ind. ml^–1) which would heavily graze on PNAN (phototrophic nanoflagellates). Top-down control can thus occur in this lake during short periods of long hydrologic residence time.     

Zooplankton induced decrease in inorganic phosphorus uptake by plankton in an oligotrophic lake

Experiments conducted on samples collected from a large oligotrophic lake revealed the following: (1) excretion rates of PO _inf4 ^sup3– by single Daphnia thorata were below detection (5 pmol animal^–1 min^–1) in 20 ml of oligotrophic lake water over a period of 10 min, (2) experimental addition of D. thorata to 20 ml aliquots of lake water decreased community-wide microbial uptake of PO _inf4 ^sup3– on two occasions (as measured by ³²PO _inf4 ^sup3– incorporation), and (3) the presence of D. thorata increased uptake by organisms smaller than 1µm, and decreased uptake by large phytoplankton. The specific mechanism for these responses remains unclear, but the results imply that when phytoplankton larger than 1µm encounter cm scale patches of water recently occupied by Daphnia they may experience decreased PO _inf4 ^sup3– availability rather than elevated concentrations of PO _inf4 ^sup3– caused by excretion. We show that ³²P uptake experiments using natural plankton assemblages can be influenced by the presence or absence of large zooplankton, and that neither grazing, turbulence, nor PO _inf4 ^sup3– excretion can account for this influence.     

Feeding the fairy shrimp Streptocephalus (Anostraca - Crustacea) with the rotifer Anuraeopsis

Laboratory-cultured Streptocephalus torvicornis were offered 8 concentrations (from 6 to 800 ind. ml^–1) of Anuraeopsis fissa for periods of 2 h 30 min. Two size classes, small (male: 14.7 mm± 1.6, female: 15.4 mm± 1.3) and large (male: 20.0 mm±2.0, female: 23.1 mm± 1.5), of S. torvicornis were used. Functional response for large S. torvicornis (both sexes) plateaued at 400 rotifers ml^–1, while in small specimens it did so at 200 prey ml^–1. Females consumed significantly more (30%) prey than males. Large males consumed maximum 4730 rotifers h^–1, females 6560 h^–1.     

Relationship between current velocity,depth and the invertebrate community in a stable river system

The relationship between invertebrate densities, current velocity and water depth was studied in the Dan River, northern Israel. Maximum current preferences ranged from 5–120 cm sec^–1, and depth preferences ranged from 5–60 cm. Thirty-five taxa of invertebrates were collected by means of colonization cages. Larval and adult stages of 3 Elmidae (Coleoptera) species were treated separately: Limnius letourneuxi, Grouvellinus caucasicus and Elmis rioloides. Differences in current preference were observed between larval and adult stages of the same species of Elmidae. Taxa were also grouped according to preference for turbulence. Wide ranges of depth and current velocity preferences were observed. Most of the taxa were found at between 80–100 cm sec^–1 and at depths of less than 30 cm. A correlation between species diversity and current velocity was established. Velocities of 60–80 cm sec^–1 contained the greatest overlap of faunal preference. The sensitivity of selected species to stream flow reduction is discussed.IES Laboratory, Department of Zoology, Hebrew University; and the Nature Reserves Authority     

The effects of silver nitrate,colchicine, cupric sulfate and genotype on the production of embryoids from anthers of tetraploid wheat (Triticum turgidum)

Anther culture of four tetraploid wheat (Triticum turgidum) genotypes was studied using ten different culture medium treatments in a randomized block design with four replicates. Each replicate consisted of 2 pots with 3 plants. Anther donor plants were grown in a greenhouse with a 16 h day/8 h night at 25°C and 15°C, respectively. The first treatment which was considered as the control, was potato 2 medium modified by adding 0.5 g l^–1 glutamine and solidified by gelrite (4 g l^–1). The nine test treatments differed from the control by addition of 3 different concentrations of silver nitrate (1, 2.5 and 5 mg l^–1), colchicine (10, 100 and 200 mg l^–1) or cupric sulfate (2,5 and 10 mg l^–1). The study of about 2000 anthers per genotype and treatment showed that both genotype and treatment affected embryoid formation. The presence of cupric sulfate (10 mg l^–1) and silver nitrate (2.5 and 5 mg l^–1) usually increased the frequency of embryoid formation in 3 genotypes out of the 4 studied. On the contrary, colchicine had a significant and enegative effect on anther culture responses for three out of the four genotypes studied. Because of the large genotype x medium interaction, it is very difficult to identify the best medium for embryo production by all genotypes studied.