Factors affecting the frequency of concealed heterokaryosis in Saccharomyces cerevisiae

In this work, studies on the phenomenon of concealed heterokaryosis that we previously detected in the saccharomycetes yeast strains were continued. New approaches to high effectiveness of isolation of cytoductants carrying the concealed nucleus were implemented, and the composition of individual concealed heterokaryons, zygotic clones, and the first zygotic buds was analyzed by a micromanipulator. The relationship between a delay in the growth of the parental strain (a potential donor of the concealed nucleus) and a decline in the frequency of the appearance of concealed heterokaryons (HKC) was observed. It is assumed that different replication rates of two nuclei of the heterokaryon probably underlie the appearance of HKC. A drastically decreased level of replication of one of the parental nuclei may be connected with the fact that binuclear buds appear extremely rarely and give rise to the rapidly "purified" progeny consisting of cells carrying the second nucleus with normal replication. A lack of the phenotype allows rare binuclear cells to persist as concealed heterokaryons. HKC may be detected only when cells of either parental type are isolated on the corresponding selective media.     

Detection of Concealed “Illegitimate” Nuclei in Tetrad Analysis of the Diploid Progeny of Heterokaryons in Saccharomyces cerevisiae

In this work, the studies on the previously detected phenomenon of concealed heterokaryosis in Saccharomyces cerevisiaewere continued. In genetic and Southern blotting experiments, one of the nuclei in the heterokaryon was shown to be active (capable of division and ensuring the corresponding cell phenotype), whereas the other was not expressed until the heterokaryotic clone was transferred to the medium selective for this concealed nucleus. Moreover, the concealed nucleus was able to assume the active state after fusion with the second parental nucleus. It was analyzed whether the nuclei with new marker combinations occurring in meiosis can behave as exceptional nuclei. Tetrad analysis of hybrids carrying the kar1mutation in their nuclei revealed the relatively high percentage of exceptional tetrads (more than 10%). One spore in these tetrads usually formed diploid cells capable of sporulation. The presented data of genetic and molecular biological studies testify in favor of the assumption that abnormal spores contain two nuclei, which form an illegitimate hybrid after fusion. An extraneous nucleus (termed x) has usually a genotype close to that of one of the spores in this tetrad. Thus, it was assumed that the additional DNA replication round occurs in the absence of cell division during one of meiotic divisions. Results of cytological analysis conducted by the method of specific DNA staining confirmed the existence of exceptional tetrads, one spore of which contains two nuclei.     

Concealed nuclei in Saccharomyces strains

We have found that cells derived from heterokaryons (HK) showing phenotypical traits, coded by the nucleus of one parental strain and by the cytoplasm of the other, may produce mitotic progeny in which the second nucleus is apparently present but not expressed. This 'concealed' nucleus can be forced to expression after growth of these cytoductants on proper selective media. Using a micromanipulator, the buds containing both parental nuclei were isolated in various crosses. Cloning these HK from a rich medium (YEPD) revealed that nearly all of them were composed of a mixture of hybrid cells and cells of one of the parents. Cells of the other parent were present in a very small proportion, if detectable at all. We showed that the percentage of concealed HK decreases when limiting the growth of the strain that serves as a donor of the concealed nucleus. Consequently, our explanation for the presence of concealed nuclei in HK is the low production rate of daughter cells containing both nuclei, which accounts for the lack of a visible phenotype in HK, together with the low replication rate (or fast nuclease degradation) of one of the nuclei. In homosexual crosses, selection allows us to switch the concealed nucleus to normal replication rate. Some abnormalities of meiosis due to hidden nuclei are shown.     

Organogenesis in Taxus brevifolia tissue cultures

Summary A method is described for multiple shoot and plantlet formation from zygotic embryos of Taxus brevifolia. Adventitious bud primordia were best induced by culturing zygotic embryos on 1/2B5 medium supplemented with 10 M BA for 14 days. Further vegetative buds were produced following subculture to half-strength McCown's basal salt medium containing 1.0% activated charcoal. Individual adventitious shoots were excised and approximately 5% of these formed roots. Rooting frequency was increased to 58% by a single treatment with ABT rooting powder. Vigorous growing Taxus brevifolia plants were established after transfer to plant growth medium.     

Quantitative,three-dimensional ultrastructure of isolated corn (Zea mays) sperm cells

Summary Five isolatedZea mays sperm cells, taken from the same population as used for a previous morphometric study, were serially ultrathin sectioned and computer-reconstructed to yield three-dimensional images as well as quantitative data. All cells were found to be essentially spherical and to contain a full complement of cell constituents except plastids and microtubules. The nuclei of three cells were highly curved into a C or V shape while the other two nuclei were not curved, but were more spherical to disc shaped. The three curved nuclei were heterochromatic in appearance, the other two were more euchromatic. Mitochondria were closely associated with the nuclei, were predominately in the form of large, variously shaped complexes, and ranged in number from 7 to approximately 74 per cell. Dictyosomes tended not to be close to the nucleus and ranged in number from 6 to 23 per cell. The endoplasmic reticulum was similarly not typically associated with the nucleus, and varied from extensive sheet-like areas to small membranous whorls. In addition to confirming the findings of previous studies on isolated corn sperm cells and providing new three-dimensional and distribution data, the results of the present work underscore the existence of a high degree of morphological variability amongZea mays sperm cells of a population.Abbreviations ER endoplasmic reticulum - SD standard deviation     

Nuclear selection in monokaryotization of dikaryotic mycelia ofPholiota nameko as described by leading and following nuclei

To examine monokaryotization of dikaryotic mycelia ofPholiota nameko, 18 monokaryotic stocks were used to produce a total of 130 dikaryotic stocks by reciprocal crossing. Monokaryotized mycelium was raised from dikaryotic mycelium in the peripheral zone of the growing colony. The stocks mated with a particular group of monokaryons produced wide-range monokaryotization at higher rates than the other combinations of hybridization. The growth rates of the monokaryotized mycelia exceeded from those of the corresponding parental dikaryons. The monokaryotized mycelium was isolated and back-crossed to parental monokaryotic stocks. Most of the isolates had nuclear types similar to only one of the parental stocks, while the replicates of isolates from two dikaryotic hybrids showed split nuclear type compositions. It is suggested that a relative dominance is active in the selection of one of the two nuclei of the dikaryotic cells in monokaryotization. The hierarchy of relative dominance among nuclei of 18 parental monokaryotic stocks in the monokaryotization of their reciprocal crossing products was estimated. We propose the involvement of a cascade process in dikaryotic cell division, in which the first dividing nucleus (to be found in the monokaryotized cell) may act as the leading nucleus and the other one as the following nucleus.     

Detection of concealed "illegitimate" nuclei in tetrad analysis of the diploid progeny of heterokaryons in Saccharomyces cerevisiae

In this work, the studies on the previously detected phenomenon of concealed heterokaryosis in Saccharomyces cerevisiae were continued. In genetic and Southern blotting experiments, one of the nuclei in the heterokaryon was shown to be active (capable of division and ensuring the corresponding cell phenotype), whereas the other was not expressed until the heterokaryotic clone was transferred to the medium selective for this concealed nucleus. Moreover, the concealed nucleus was able to assume the active state after fusion with the second parental nucleus. It was analyzed whether the nuclei with new marker combinations occurring in meiosis can behave as exceptional nuclei. Tetrad analysis of hybrids carrying the kar1 mutation in their nuclei revealed the relatively high percentage of exceptional tetrads (more than 10%). One spore in these tetrads usually formed diploid cells capable of sporulation. The presented data of genetic and molecular biological studies testify in favor of the assumption that abnormal spores contain two nuclei, which form an "illegitimate" hybrid after fusion. An extraneous spore (termed x) has usually a genotype close to that of one of the spores in this tetrad. Thus, it was assumed that the additional DNA replication round occurs in the absence of cell division during one of meiotic divisions. Results of cytological analysis conducted by the method of specific DNA staining confirmed the existence of exceptional tetrads, one spore of which contains two nuclei.     

Effects of plant growth regulators on secondary wall thickening of cotton fibres

The effect of plant growth regulators on the secondary wall thickeningof cotton fibre was studied. The results indicated that the GA_S andiP+iPA levels in the fibre of field-grown cotton plantsremained almost constant but the IAA and ABA levels changed considerably duringfibre development. Although the change in both IAA and ABA levels seemed not tobe closely related with the rate of cellulose accumulation, there was still arelationship between the ratio of ABA to IAA and secondary wall thickening. Inin vitro studies, ABA (50mol·L^–1) markedly enhanced theaccumulation of dry matter and cellulose in the fibre cell wall duringsecondarywall thickening, but no similar effect was observed with NAA, GA₃ orkinetin treatments. The role of ABA in secondary wall thickening of cottonfibreis discussed.     

Formation of the Interaction between the Wave Components of the Main EEG Rhythms in Children up to Five Years of Age

Using the analysis of the interaction between EEG components [1], the authors studied the regularities of the formation of the EEG wave structure in 36 children aged 4 to 7 months, 2 to 3 years, and 4 to 5 years. The EEG of 4- to 7-month-old children had a relatively organized temporal structure, whose components were connected mainly with those of the slow-wave range. This structure is reproduced in most of the leads and is more stable in the anterior cortical areas than in the posterior ones. The waking 2- to 5-year-old children had two functional nuclei in the relation structure of the EEG components characterized by statistically significant enhancement in the probability of the consecutive appearance of waves of certain ranges. These nuclei include a slow-wave nucleus in the range of the ₂–₁-frequencies and an alpha-nucleus in the ₁- and ₂-ranges (₂ and ₁ 2- to 3-year-old children) of the EEG frequencies. The relations between the components of the slow-wave nucleus begin to form in infants, prevail in 2- to 3-year-old children, and weaken by the age of 4 to 5 years. A trend is observed in 2- to 5-year-old children towards an increase in the average frequency of the waves that form the slow-wave nucleus. The temporal (strengthening interaction between the waves) and spatial organization of the interaction between the wave components of the alpha-nucleus is accelerated at the age of 4 to 5 years. The average frequency of the components forming this nucleus gradually increases with the children's age from 4 to 7 months to 4 to 5 years. Competitive relations exist between the two nuclei, characterized by a significant decrease in the probability of transitions between the components of different nuclei. The ₂-component may play a special role of the connecting link between the nuclei at the age of 4 to 5 years, the interwave transition from the - to -frequencies and from the latter to the -range being effected by a leaplike rather than smooth frequency increase. The character of local and spatial rearrangements of the EEG temporal structure detected in this study reflects a gradual morphofunctional maturation of the brain as a system that maintains the specific features of self-regulation mechanisms and coordination of the intersystem interactions at various stages of a child's development.     

The arrangement of chromosomes in nuclei of sperm from plethodontid salamanders

Plethodontid salamanders have n = 13 or 14 large metacentric or sub-metacentric chromosomes. Sperm nuclei from Plethodon cinereus measure 72×1 m. The nucleoprotein of spermatids is at first finely granular. In elongate spermatids it clumps into larger granules, which then fuse to form the compact nucleoprotein of the mature sperm. The nuclei of mature sperm are negatively birefringent with respect to their length. — ³H RNA complementary to high-density satellite DNA of centromeric heterochromatin in P. cinereus has been hybridized in-situ to spermatids and sperm, and its site of binding to these cells has been examined by autoradiography. Labelling of round spermatid nuclei is localized in a single patch. Elongate spermatid nuclei are labelled only over the rear quarter of the nucleus. Label over the nuclei of mature sperm is localized in a region extending 10–20 m forwards from the rear of the nucleus. — In P. cinereus the ribosomal genes are located near the centromere on the short arm of chromosome 7. ³H ribosomal RNA hybridizes to a single patch in round spermatid nuclei. Elongate spermatid nuclei show label over a short segment of the rear half of the nucleus. In spermatids nearing maturity the labelled region is never more than 20 m long. — These results indicate that in P. cinereus each chromosome is arranged in a U formation with its centromere at the base of the sperm nucleus, and its arms extended forwards along the length of the nucleus. — Among plethodontids, increase in C value and corresponding increase in chromosome size is accompanied by increase in the length rather than the width of the sperm nucleus. — ³H ribosomal RNA hybridizes to a short segment in spermatid and sperm nuclei from Xenopus and Triturus. In these animals, the position of the labelled segment varies from sperm to sperm.     

Pulse schemes for the measurement of3 JC′Cγ and3 JNCγ scalar couplings in 15N,13C uniformly labeled proteins

Pulse sequences are presented for the measurement of³J_CC and³J_NC scalar couplings for allC containing residues in¹⁵N,¹³C uniformly labeled proteins. The methodsdescribed are based on quantitative J correlation spectroscopy pioneered byBax and co-workers [Bax et al. (1994) Methods Enzymol., 239, 79–105].The combination of ³J_CC and³J_NC scalar coupling constants allows theassignment of discrete rotameric states about the ₁ torsion angle in cases where such states exist or, alternatively,facilitates the establishment of noncanonical ₁conformations or the presence of rotameric averaging. The methods areapplied to a 1.5 mM sample of staphylococcal nuclease.     

Vertebrate-like βγ-crystallins in the ocular lenses of a copepod

The diverse crystallins are water-soluble proteins that are responsible for the optical properties of cellular lenses of animal eyes. While all vertebrate lenses contain physiological stress-related - and -crystallins, some also contain taxon-specific, often enzyme-related crystallins. To date, the - and -crystallins have been found only in vertebrate lenses. Here we report lenses from an invertebrate, the pontellid copepod Anomalocera ornata, accumulate -crystallin family members as judged by immunocytochemistry, western immunoblotting and microsequencing. Our data provide the first example of -crystallin members in an invertebrate lens, establishing that the use of this protein family as lens crystallins is not confined to vertebrates.     

Cytoarchitectonic pattern of the hypothalamus in the cobra,Naja naja

Summary The distribution and cytoarchitectonic pattern of the magno- and parvocellular hypothalamic nuclei of the cobra, Naja naja, are described at the light-microscopic level. With respect to their tinctorial affinity to paraldehyde fuchsin (AF) as a representative of the Gomori-type of stains, the magnocellular neurons belong to the AF-positive and the parvocellular neurons to the AF-negative elements. In addition to the supraoptic and paraventricular nuclei proper, two accessory aggregations of magnocellular neurons, the nucleus retrochiasmaticus and nucleus circularis, can be identified. Although in a peculiar location, they may be regarded as subunits of the supraopticoparaventricular neurosecretory complex. As many as 22 AF-negative nuclear areas are identified in the hypothalamus of the cobra. The nucleus periventricularis hypothalami of earlier authors is subdivided into several circumscribed neuronal complexes. The nucleus arcuatus, nucleus hypothalamicus lateralis and nucleus lateralis recessus infundibuli are well developed. An attempt is made to interpret the significance of these nuclei on a comparative and phylogenetic basis.On leave from the Department of Zoology, Nagpur University, Nagpur, India     

Induction of programmed cell death in human retinoblastoma Y79 cells by C2-ceramide

C₂-ceramide, a cell-permeable analogue of ceramide, induced significant, dose- and time-dependent death in human retinoblastoma Y79 cells. Dying cells strongly displayed the morphology of apoptosis as characterized by microscopic evidence of cell shrinkage, membrane blebbing, nuclear and chromatin condensation and degeneration of the nucleus into membrane-bound apoptotic bodies. Upon induction of apoptosis Y79 cells evidence early phosphatidylserine externalization, as shown by annexin V-FITC. Apoptosis was also assessed by monitoring changes in cell granularity by staining with the combined fluorescent dyes acridine orange and ethidium bromide. C₂-ceramide induced these morphological changes without a concomitant production of oligonucleosomal fragments responsible for the DNA ladder and without changes in p53 protein level. Apoptosis was accompanied by accumulation of a modified Bcl-2 protein with a slower-mobility form, and by proteolytic cleavage of PARP. The effect seemed to be specific for C₂-ceramide, as C₂-dihydroceramide, or other amphiphilic lipid analogues, or products of ceramide hydrolysis were ineffective. The effect also depended on mRNA and protein synthesis as it was markedly inhibited by actinomycin D and cycloheximide. Sphingomyelinase and interleukin-l, which are known to activate the sphingomyelin turnover leading to ceramide generation, also induced apoptosis mimicking the effects of ceramide. These findings propose ceramide as an activator of the suicidal program in Y79 cells.     

The effectiveness of nitrogen sources in Feijoa somatic embryogenesis

Immature and mature zygotic embryos excised from Feijoa fruits were employed as explants and the effects of NH₄ ⁺ and NO₃ ^– ionic concentration in basal LPm culture medium supplemented with 2,4-D (10 M) were evaluated. Moreover, the addition of 4 mM of Asn, Gln, and Arg, and levels of Gln (0 to 8 mM) were tested. The original NH₄ ⁺ and NO₃ ^– concentration present in the LPm culture medium supplemented with Gln (4 mM) resulted in the highest somatic embryo number from immature zygotic embryos. For mature zygotic embryos, the addition of Asn, Gln or Arg to the basal LPm culture medium resulted in improved somatic embryogenesis induction. Ten weeks in culture allowed the highest somatic embryo number when mature zygotic embryos were used as explant. Half-strength MS culture medium supplemented with BAP (0.5 M) enhanced the conversion of somatic embryos to plantlets.     

Location of heitz's zerstäubungsstadium (dispersion phase) in the mitotic cycle ofPhaseolus coccineus and the concept of Angiosperm endomitosis

Summary DNA microdensitometry and autoradiography after treatment with³H-thymidine were used to study the phase of dispersion of chromocenters (Z phase) in parallel with chromocentric nuclei inPhaseolus coccineus. In all materials studied, two types of chromocentric nuclei were present.In radicle apices of dry seeds, two classes of nuclear DNA contents were measured, 2 C (G₁) and 4 C (G₂). The 2 C DNA class comprised all chromocentric type I nuclei, the 4 C class included Z phases and chromocentric type II nuclei. The 4 C (G₂) condition of Z phases implies that Z phases maintain their nuclear structure for some time after the end of DNA replication. Shoot apices also contain 2 C (G₁) and 4 C (G₂) nuclei but 4 C nuclei (Z phases and chromocentric type II nuclei) are rare.In seedling root apices, Z phases are from 1.02 to 4.08 times as frequent as prophases. This excludes that Z phase is a very early prophase. DNA microdensitometry shows that the chromocentric type I includes 2 C (G₁) nuclei and nuclei in the first part of the S phase, Z phases include 4 C (G₂) nuclei and nuclei in the last stage of the S phase and chromocentric type II includes mainly 4 C (G₂) nuclei and nuclei in the second part of S. After 90 minutes of treatment with³H-thymidine all Z phase nuclei are labeled. This result and the microdensitometric data unequivocally demonstrate that Z phase is located at the end of S.The present results and those of previous authors on Z phase are discussed in relation to Geitler's concept of Angiosperm endomitosis. It is concluded that the term Angiosperm endomitosis must be abandoned and substituted by the term chromosome endoreduplication.     

Low temperature optical spectroscopy of cobalt-substituted hemocyanin from Carcinus maenas

In this work we report the optical absorption spectra of three cobalt-substituted derivatives of hemocyanin (He) from Carcinus maenas, in the temperature range 300–20 K. The derivatives studied are the mononuclear (Co²⁺)-He with a single cobalt ion in the Cu_A site, the binuclear (Co²⁺)₂-He and the binuclear mixed metal (Co²⁺-Cu¹⁺)-He. At low temperature three main bands are clearly resolved; the temperature dependence of their zeroth, first and second moments sheds light on the stereodynamic properties in the surroundings of the chromophore. Within the limits of the reported analysis, in the binuclear derivatives the motions coupled to the chromophore appear to be essentially harmonic in the whole temperature range investigated; moreover the data are consistent with the presence of an exogenous ligand strongly bound to the two metal ions. For the mononuclear derivative an essentially harmonic behavior is evident only up to 200 K where the data are consistent with the presence of an exogenous ligand much less strongly bound, while at higher temperatures the behavior of the spectra indicates the onset of very large anharmonic contributions to motions, that plausibly involve the above exogenous ligand and, quite likely, the entire active site.Abbreviations He Hemocyanin - M₀ zeroth moment - M₁ first moment - M₂ second moment - (Co^2–)₂-He binuclear bicobalt hemocyanin derivative - (Co²⁺)-He mononuclear monocobalt hemocyanin derivative - (Co²⁺-Cu¹⁺)-He binuclear mixed metals hemocyanin derivative - LFT ligand field theory - CT charge transfer - EPR electronic paramagnetic resonance - XANES X-ray absorption near edge structure Correspondence to: L. Cordone     

Genetic regulation of the replication pattern of polytene chromosomes in interspecific hybrids of Drosophila

The sixth chromosome (microchromosome) of D. littoralis changed its replication pattern in nuclei of the salivary gland cells in reciprocal F₁ hybrids between D. virilis and D. littoralis. — To locate the factor (or factors) in the D. virilis chromosomes, which may influence the replication pattern of the sixth chromsome of D. littoralis, we produced hybrid stocks with synthetic karyotypes characterized by different combinations of D. littoralis homologous chromosomes and hybrid chromosomes. Based on autoradiographical studies of DNA synthesis in synthetic karyotypes, it may be concluded that the dominant factor (or factors), which influences the replication of the sixth chromosome of D. littoralis, is located on the homeologous microchromosomes of D. virilis. The possible interrelation between the changed replication pattern of D. littoralis sixth chromosome and its atypical behaviour at early embryogenesis in (D. virilis x D. littoralis) F₁ hybrids is discussed.     

The αβ complexes of ATP synthase: the α3β3 oligomer and α1β1 protomer

The basic structures of the catalytic portion (F₁, ₃₃) of ATP synthase are the ₃₃ hexamer (oligomer with cooperativity) and ₁₁ heterodimer (protomer). These were reconstituted from the and subunits of thermophilic F₁ (TF₁), and the ₃₃ hexamer was crystallized. On electrophoresis, both the dimer and hexamer showed bands with ATPase activity. Using the dimer and hexamer, we studied the nucleotide-dependent rapid molecular dynamics. The formation of the hexamer required neither nucleotide nor Mg. The hexamer was dissociated into the dimer in the presence of MgADP, while the dimer was associated into the hexamer in the presence of MgATP. The hexamer, like mitochondrial F₁ and TF₁, showed two kinds of ATPase activity: one was cooperative and was inhibited by only one BzADP per hexamer, and the other was inhibited by three BzADP per hexamer.