Karyotypanalyse an Hand des Färbungsmusters der Metaphasechromosomen von Cypripedium debile und Trillium kamtschaticum

The somatic metaphase chromosomes of Cypripedium debile and Trillium kamtschaticum are stained differentially by treatment with an acetic orcein-hydrochloric acid mixture: In Cypripedium, the heterochromatic segments stain densely whereas the euchromatic segments are unstained. In Trillium, in contrast, the heterochromatic segments are unstained and euchromatin is stained. Such an inconsistency in stain patterns is considered to stem from different species-specific reactivity of heterochromatin and euchromatin to hydrochloric acid in the staining medium.—In metaphase chromosomes of Cypripedium stained with fast green (Alfert and Geschwind, 1953), the euchromatic segments are stained positively, whereas the heterochromatin, as well as the chromocenters, are unstained. In Trillium, all heterochromatin, euchromatin and chromocenter are stained homogeneously by this method. These results indicate that the heterochromatic segments and chromocenters in Cypripedium are associated with non-histon type proteins, whereas the euchromatic segments in Cypripedium, as well as two different types of the segments and the chromocenters in Trillium, are all bound to histon type proteins. — From these findings, it is concluded that two types of heterochromatin occur in plant materials, though it remains unsettled whether they correspond to - and -heterochromatin as found in Dipteran giant chromosomes.—Karyotype analysis is made on the basis of the differential staining pattern of chromosomes revealed by means of acetic orcein-hydrochloric procedure.     

Accumulation and translocation of sulphate in excised maize roots as affected by different saline concentrations in the outer medium

Abstract

Accumulation and translocation of sulphate in excised maize roots, submerged in rising saline concentrations, were investigated. It was shown that the accumulation of sulphate is not depressed by concentrations from 1 to 50 mM of NaCl or KCl, it is weakly increased by concentrations of the same salts 100 mM and it is gradually lowered by concentrations from 1 to 100 mM of MgCl₂.

On the contrary the translocation is gradually inhibited by rising concentrations of NaCl, KCl and MgCl₂. A 100 mM NaCl concentration considerably loweres the translocation in 24 hours, but does not affect accumulation. Accumulation and translocation are strongly depressed by the inhibitors of oxydative phosphorylation (2,4 DNP or CCCP) and by 200 mM NaCl, KCl or MgCl₂ concentrations.

It is concluded that accumulation and translocation are active processes as they are reduced by 2,4 DNP or CCCP; that the small increase in accumulation observed by 100 mM NaCl or KCl concentration is due probably to the discharging action of cations exercited on the membranes of root cells and that only the second step of ion translocation, i.e. ion secretion in xylem, is sensible to the presence of high saline concentrations of NaCl or KCl in the outer medium.     

Early and late DNA replication in respectively condensed and decondensed heterochromatin ofAllium carinatum

Summary The structure and ultrastructure of nuclei in the S period and other phases of the mitotic cell cycle have been studied in semi- and ultrathin sections of root tips ofAllium carinatum. Significant structural differences have been found and classified by means of DNA measurements by scanning photometry of Feulgen-stained squash preparations. In G₁ and early S (S₁ and S₂) the euchromatin forms small, compact and electron-dense patches, while the heterochromatin is condensed into a number of chromocenters of the same electron-density as the euchromatin. In middle S (S₃) the euchromatic elements become larger and more thread-like. In late S (S₄) the euchromatin appears in the form of thick and uniform strands as in G₂, and the heterochromatin decondenses into strands of the same, or a little higher, diameter, as the euchromatin. DNA replication starts in the condensed heterochromatin (S₁, becomes shifted to euchromatin (S₂), continues over both eu- and heterochromatin during middle S (S₃), and is restricted to decondensed heterochromatin in late S (S₄). Quantitative data of various nuclear parameters are given for the different stages. The results are discussed in relation to the species-specific nuclear ultrastructure, its molecular basis, and its variation during the mitotic interphase, as well as with respect to the timing and structural expression of DNA replication.     

Aggregation of mono- and dinucleosomes into chromatin-like fibers

Three classes of chicken erythrocyte chromatin particles differing in their content of lysine-rich histones and/or spacer DNA have been studied in order to determine their ability to aggregate into complexes resembling those observed in native chromatin. The complexes have been obtained in the presence of MgCl₂ and NaCl and studied by electron microscopy. Mononucleosomes, containing spacer DNA and histones H1 and H5, give rise to thick (about 70 nm) ellipsoidal particles in the presence of 0.5 mM MgCl₂. These particles are disrupted by the addition of small amounts of NaCl (5–20 mM). On the other hand in 0.5 mM MgCl₂ dinucleosomes give rise to regular fibrous complexes of about 40 nm in diameter which are very similar to native chromatin fibers. These complexes are much more stable when NaCl is added. We conclude that for the stability of nucleosomal aggregates, similar to native chromatin fibers, a continuity of DNA structure is not required, but the presence of divalent cations, spacer DNA and lysine-rich histones is essential.     

The Paf1 complex factors Leo1 and Paf1 promote local histone turnover to modulate chromatin states in fission yeast

The maintenance of open and repressed chromatin states is crucial for the regulation of gene expression. To study the genes involved in maintaining chromatin states, we generated a random mutant library in Schizosaccharomyces pombe and monitored the silencing of reporter genes inserted into the euchromatic region adjacent to the heterochromatic mating type locus. We show that Leo1–Paf1 [a subcomplex of the RNA polymerase II‐associated factor 1 complex (Paf1C)] is required to prevent the spreading of heterochromatin into euchromatin by mapping the heterochromatin mark H3K9me2 using high‐resolution genomewide ChIP (ChIP–exo). Loss of Leo1–Paf1 increases heterochromatin stability at several facultative heterochromatin loci in an RNAi‐independent manner. Instead, deletion of Leo1 decreases nucleosome turnover, leading to heterochromatin stabilization. Our data reveal that Leo1–Paf1 promotes chromatin state fluctuations by enhancing histone turnover.     

Control of puffing activity in three chromosomal segments of explanted salivary gland cells of Chironomus thummi by variation in extracellular Na+,K+ and Mg2+

Salivary glands of Chironomus thummi larvae were incubated in media composed of those $\text{NaCl}\text{KCl}\text{,}\text{MgCl}{}_2\text{NaCl}\text{or}\text{MgCl}{}_2\text{KCl}$ combinations and at concentrations which they tolerated without visible damage. Resulting changes in puffing activity were recorded for three chromosomal segments. Within certain combinatorial ranges $\text{NaCl}\text{KCl}\text{,}\text{MgCl}{}_2\text{NaCl}\text{and}\text{MgCl}{}_2\text{KCl}$ induced puffs in the three segments. Each inducing range is depicted as a ‘puff-inducing field’ for extracellular ion concentrations (IF^e) in a two-dimensional lattice. The IF^e_s are coherent, distinctly delineated and highly overlapping. At most places a transition from 0 to 100 % puff induction (induction of respective puff in each nucleus) depends on changes in media composition of 10–20 mM. Ion sensitivities of the three chromosomal segments were computed for NaCl/KCl/MgCl₂ combinations and were found to conform to actual puff-inducing capacities of selected NaCl/KCl/MgCl₂ media.     

Early replicating DNA in heterochromatin ofPlagiochila ovalifolia (Liverworts)

The timing of DNA replication of heterochromatin in malePlagiochila ovalifolia was investigated by the use of³H-thymidine autoradiography. The estimated duration of the mitotic cycle was as follows: S period, 19 hr: G₂+prophase, 10 hr; G₁+meta-, ana-, telophase, 5 hr; total mitotic cycle, 34 hr. The first appearance of silver grains over the chromosomes was observed at 8 hr after the beginning of pulse labelling at which time the silver grains were only over the euchromatic regions, not over the heterochromatic regions. This labelling pattern was also observed at 10 to 15 hr. The heterochromatic regions having more grains than the euchromatic regions were observed at 20 to 25 hr. These results show that the DNA of the heterochromatin of this species is replicated earlier than the euchromatin.     

Benzamide-inhibitable alterations in spectral properties of chromatin accompany DNA repair in UVC-exposed L5178Y cells

Summary We examined the response of chromatin to increasing NaCl and MgCl₂ concentrations in UVC-irradiated L5178Y (LY) R and S cells, using the spectral index method (Dixon and Burkholder 1985). We have found an alteration in chromatin properties 1 h after UVC-irradiation of repair proficient LY-S cells, but no change in repair deficient LY-R cells. The change was shown as lowered spectral index, indicating that at given Na⁺ and Mg⁺⁺ concentrations (1 or 200 mM NaCl, 0 or 0.5 mM MgCl₂) chromatin from UVC-irradiated LY-S cells was more compact than that from unirradiated ones. Benzamide treatment reversed the effect of UVC-irradiation in LY-S cells and did not change the response pattern of chromatin from LY-R cells or unirradiated LY-S cells.     

Binding to the high-affinity substrate site of the (Na+ + K+)-dependent ATPase

The (Na⁺ + K⁺)-dependent ATPase exhibits substrate sites with both high affinity (K _m near 1 µM) and low affinity (K _m near 0.1 mM) for ATP. To permit the study of nucleotide binding to the high-affinity substrate sites of a canine kidney enzyme preparation in the presence as well as absence of MgCl₂, the nonhydrolyzable - imido analog of ATP, AMP-PNP, was used in experiments performed at 0–4°C by a centrifugation technique. By this method theK _D for AMP-PNP was 4.2 µM in the absence of MgCl₂. Adding 50 µM MgCl₂, however, decreased theK _D to 2.2 µM; by contrast, higher concentrations of MgCl₂ increased theK _D until, with 2 mM MgCl₂, theK _D was 6 µM. The half-maximal effect of MgCl₂ on increasing theK _D occurred at approximately 1 mM. This biphasic effect of MgCl₂ is interpreted as Mg²⁺ in low concentrations favoring AMP-PNP binding through formation at the high-affinity substrate sites of a ternary enzyme-AMP-PNP-Mg complex; inhibition of nucleotide binding at higher MgCl₂ concentrations would represent Mg²⁺ acting through the low-affinity substrate sites. NaCl in the absence of MgCl₂ increased AMP-PNP binding, with a half-maximal effect near 0.3 mM; in the presence of MgCl₂, however, NaCl increased theK _D for AMP-PNP. KCl decreased AMP-PNP binding in the presence or absence of MgCl₂, but the simultaneous presence of a molar excess of NaCl abolished (or masked) the effect of KCl. ADP and ATP acted as competitors to the binding of AMP-PNP, although a substrate for the K⁺-dependent phosphatase reaction also catalyzed by this enzyme,p-nitrophenyl phosphate, did not. This lack of competition is consistent with formulations in which the phosphatase reaction is catalyzed at the low-affinity substrate sites.     

Assembly of nucleosomal DNA in a cell-free extract from wild-type and top1− strains ofUstilago maydis

An in vitro nucleosome assembly system has been established from cell-free extracts of the fungusUstilago maydis. The extract catalyzed DNA supercoiling in the absence of exogenously added co-factors such as ATP and MgCl₂ and was inhibited by moderate concentrations (200 mM) of KCl or NaCl. DNA supercoiling occurs via the formation of nucleosomes. Similar extracts, displaying the same activity, were prepared fromSaccharomyces cerevisiae andCandida albicans, suggesting that the extract preparation protocol may be useful for many lower eukaryotic systems. An extract prepared from a strain ofU. maydis lacking topoisomerase I failed to catalyze nucleosome assembly, clearly implicating this enzyme in this process. Addition of purified topoisomerase I, and, to a lesser extent, topoisomerase II, to the top1^? extract regenerated the supercoiling activity. Our results provide a method for preparing assembly extracts from organisms, that are particularly amenable to genetic manipulation.     

Assembly of nucleosomal DNA in a cell-free extract from wild-type and top1? strains ofUstilago maydis

An in vitro nucleosome assembly system has been established from cell-free extracts of the fungusUstilago maydis. The extract catalyzed DNA supercoiling in the absence of exogenously added co-factors such as ATP and MgCl₂ and was inhibited by moderate concentrations (200 mM) of KCl or NaCl. DNA supercoiling occurs via the formation of nucleosomes. Similar extracts, displaying the same activity, were prepared fromSaccharomyces cerevisiae andCandida albicans, suggesting that the extract preparation protocol may be useful for many lower eukaryotic systems. An extract prepared from a strain ofU. maydis lacking topoisomerase I failed to catalyze nucleosome assembly, clearly implicating this enzyme in this process. Addition of purified topoisomerase I, and, to a lesser extent, topoisomerase II, to the top1^– extract regenerated the supercoiling activity. Our results provide a method for preparing assembly extracts from organisms, that are particularly amenable to genetic manipulation.     

Changes in Chromosomal Localization of Heterochromatin-binding Proteins during the Cell Cycle in Drosophila

We examined the heterochromatic binding of GAGA factor and proliferation disrupter (Prod) proteins during the cell cycle in Drosophila melanogaster and sibling species. GAGA factor binding to the brown^Dominant AG-rich satellite sequence insertion was seen at metaphase, however, no binding of GAGA factor to AG-rich sequences was observed at interphase in polytene or diploid nuclei. Comparable mitosis-specific binding was found for Prod protein to its target satellite in pericentric heterochromatin. At interphase, these proteins bind numerous dispersed sites in euchromatin, indicating that they move from euchromatin to heterochromatin and back every cell cycle. The presence of Prod in heterochromatin for a longer portion of the cell cycle than GAGA factor suggests that they cycle between euchromatin and heterochromatin independently. We propose that movement of GAGA factor and Prod from high affinity sites in euchromatin occurs upon condensation of metaphase chromosomes. Upon decondensation, GAGA factor and Prod shift from low affinity sites within satellite DNA back to euchromatic sites as a self-assembly process.     

Differential reaction of condensed and diffuse chromatin to polyamines. II. Effect of putrescine on the chromatin of mitotic chromosomes]

The response of euchromatin and heterochromatin to putrescine was studied using chinese hamster mitotic chromosomes with heterochromatic segments delayed in condensation due to BUdR treatment. These heterochromatic segments did not react to the condensing effect of putrescine looking during metaphase still more elongated. Additional decondensed segments occurred in chromosomes. This is interpreted as the condensing effect of putrescine on euchromatic segments which accelerates transition of the cells into metaphase with preserved BUdR-induced chromosomal decondensation.     

Artificial fertilization of gametes from the South African clawed frog,Xenopus laevis

A reproducible and effective method for fertilization eggs of Xenopus laevis was developed based of systematic manipulation of environmental factors. The effects of varying concentrations of individual components of a fertilization medium were tested by measuring jelly swelling, sperm motility, and sperm longevity. Results were used to develop an improved medium for fertilization, consisting of 41.25 mM NaCl, 1.25 mM KCl, 0.25 mM CaCl₂, 0.0625 mM MgCl₂, 0.5 mM Na₂HPO₄, 2.5 mM HEPES, 1.9 mM NaOH, final pH(2°) 7.8.     

Enantioselective Bioaccumulation of Diniconazole in Tenebrio Molitor Larvae

The enantioselective bioaccumulation of diniconazole in Tenebrio molitor Linne larva was investigated with liquid chromatography tandem mass spectrometry based on the ChiralcelOD‐3R[cellulose tri‐(3,5‐dimethylphenyl carbamate)] column. In this study we documented the effects of dietary supplementation with wheat bran contaminated by racemic diniconazole at two dose levels of 20 mg kg^‐1 and 2 mg kg^‐1 (dry weight) in Tenebrio molitor. The results showed that both doses of diniconazole were taken up by Tenebrio molitor rapidly in the first few days, the concentrations of R‐enantiomer and S‐enantiomer at high doses reached the highest level of 0.55 mg kg^‐1 and 0.48 mg kg^‐1, respectively, on the 1^st d, and the concentrations of them obtained a maxima of 0.129 mg kg^‐1 and 0.128 mg kg^‐1 at low dose, respectively, on the 3^rd d, which means that the concentration of diniconazole was proportional to the time of achieving the highest accumulated level. It afterwards attained equilibrium after a sharp decline at both 20 mg kg^‐1 and 2 mg kg^‐1 of diniconazole. The determination results from the feces of Tenebrio molitor demonstrated that the extraction recovery (ER) values of the high dose group were higher than that of the low dose group and the values were all above 1; therefore, it could be inferred that enantiomerization existed in Tenebrio molitor. Additionally, the biota accumulation factor was used to evaluate the bioaccumulation of diniconazole enantiomers, showing that the bioaccumulation of diniconazole in Tenebrio molitor was enantioselective with preferential accumulation of S‐enantiomer. Chirality 25:917–922, 2013. © 2013 Wiley Periodicals, Inc.     

Vanadate inhibition of brain (Ca+Mg)-ATPase

Vanadate was a potent inhibitor of the membrane-bound (Ca+Mg)-ATPase from rat brain, the concentration required for 50% inhibition under conditions optimal for enzymatic activity being 3 M. Vanadate inhibition increased with the MgCl₂ concentration, half-maximal inhibition occurring at 2 mM MgCl₂, near the MgCl₂ concentration required for half-maximal activation of the ATPase activity. MnCl₂ could substitute for MgCl₂, and at concentrations of 1 mM (Ca+Mn)-ATPase activity was greater than (Ca+Mg)-ATPase activity, although sensitivity to vanadate was less. Vanadate inhibition increased also with the KCl concentration, half-maximal inhibition occurring at 8 mM, again near the concentration required for half-maximal activation of ATPase activity. By contrast, NaCl stimulated (Ca+Mg)-ATPase activity without potentiating vanadate inhibition. These effects of cations on ATPase activity and vanadate inhibition resemble properties of certain transport ATPases and thus suggest mechanistic and functional similarities.     

Nucleosomal arrays self‐assemble into supramolecular globular structures lacking 30‐nm fibers

The existence of a 30‐nm fiber as a basic folding unit for DNA packaging has remained a topic of active discussion. Here, we characterize the supramolecular structures formed by reversible Mg²⁺‐dependent self‐association of linear 12‐mer nucleosomal arrays using microscopy and physicochemical approaches. These reconstituted chromatin structures, which we call “oligomers”, are globular throughout all stages of cooperative assembly and range in size from ~50 nm to a maximum diameter of ~1,000 nm. The nucleosomal arrays were packaged within the oligomers as interdigitated 10‐nm fibers, rather than folded 30‐nm structures. Linker DNA was freely accessible to micrococcal nuclease, although the oligomers remained partially intact after linker DNA digestion. The organization of chromosomal fibers in human nuclei in situ was stabilized by 1 mM MgCl₂, but became disrupted in the absence of MgCl₂, conditions that also dissociated the oligomers in vitro. These results indicate that a 10‐nm array of nucleosomes has the intrinsic ability to self‐assemble into large chromatin globules stabilized by nucleosome–nucleosome interactions, and suggest that the oligomers are a good in vitro model for investigating the structure and organization of interphase chromosomes.     

NOR associations with heterochromatin

Associations between nucleolus organizer regions (NORs) and non-acrocentric chromosomes were scored in 2,800 metaphase spreads from PHA-stimulated lymphocyte cultures (48 h) from 14 individuals. The preparations were both silver stained and C-banded. In order to calculate the expected values for associations, the ratio of heterochromatin length to euchromatin length was established for each subject. Individual C-band lengths and centromeric lengths were also determined. When silver connective (SC) associations with heterochromatin were compared to SC associations with euchromatin, the number of associations with heterochromatin was significantly greater than expected (P less than 0.000001) for each subject. The SC associations were not distributed randomly over the heterochromatin of the non-acrocentrics. Chromosomes 1 and 2 had significantly more than expected. Chromosomes 17, 18, 19, 20, and the Y had fewer than expected. NOR associations with euchromatic segments also showed a nonrandom pattern of distribution.     

Distorted heterochromatin replication in <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> polytene chromosomes as a result of euchromatin-heterochromatin rearrangements

Studies of the position effect resulting from chromosome rearrangements in Drosophila melanogaster have shown that replication distortions in polytene chromosomes correlate with heritable gene silencing in mitotic cells. Earlier studies mostly focused on the effects of euchromatin-heterochromatin rearrangements on replication and silencing of euchromatic regions adjacent to the heterochromatin breakpoint. This review is based on published original data and considers the effect of rearrangements on heterochromatin: heterochromatin blocks that are normally underrepresented or underreplicated in polytene chromosomes are restored. Euchromatin proved to affect heterochromatin, preventing its underreplication. The effect is opposite to the known inactivation effect, which extends from heterochromatin to euchromatin. The trans-action of heterochromatin blocks on replication of heterochromatin placed within euchromatin is discussed. Distortions of heterochromatin replication in polytene chromosomes are considered to be an important characteristic associated with the functional role of the corresponding genome regions.