The Development of Male and Female Regenerants by In Vitro Androgenesis in Dioecious Plant Melandrium album

We examined induced androgenesis in vitro in the dioecious plantMelandrium album and aimed to produce complete plants from culturedimmature microspores. Flow cytometric analysis of nuclear DNAcontent was used to screen ploidy levels in regenerated plantsand to estimate the nuclear genome size in plants differingin sex. Haploid and spontaneous dihaploid (polyhaploid) femalesdominated among androgenic regenerants. Androgenic males occurredsporadically. They were exclusively dihaploid and geneticallysupermales (AAYY). The progenies obtained as a result of thecrosses between supermales and standard females contained onlymales. This is the first report on complete androgenesis inM. album from the microspores carrying the Y chromosome.Copyright1994, 1999 Academic Press Melandrium album (Miller) Garcke, pollen androgenesis, sex, female, male, supermale, flow-cytometry, nuclear genome size     

Quantitative karyology of some species ofLuzula

The dimensions of metaphase chromosomes and nuclear DNA contents were measured in eight species ofLuzula. The 2 C DNA contents ranged from 8.51 pg inL. purpurea to 0.55 pg inL. pilosa. Total chromosome volume shows a linear relationship with DNA content; however, the total chromosome length of the complement of the different species is approximately constant. Nucleolar volume and the number of chromocentres in the different species also show a relationship with DNA content. Taken together, these data suggest that while chromosome fragmentation could have generated the present-day range of chromosome numbers in the genus, there have also been changes in the total quantity of DNA with the result that species with similar chromosome numbers have different DNA contents. The relationships of DNA content with chromosome volume inLuzula and other genera are compared and the differences discussed.     

Activities of DNA polymerases and RNA polymerases detected in situ in growing and differentiating cells of root cortex

Summary Activities of DNA polymerases and RNA polymerases were studied by autoradiographic methods in growing and differentiating root cortex cells ofZea mays — a species in which endomitosis occurs — andTulipa kaufmanniana — in which this process does not occur. InTulipa kaufmanniana, the highest activity of DNA polymerase appears in the nuclei of meristematic zone during the S phase of the cell cycle. InZea mays, endomitotic replication of DNA occurs in all growth and differentiation zones and the activity of DNA polymerase in the nuclei is similar to that in the meristematic zone. In both species, nuclear RNA synthesis, measured with³H uridine incorporation, is highest in the meristematic zone and declines steadily with development. Activity of nuclear RNA polymerase is present in all developmental zones in both species and is similar to that in the meristematic zone.³H uridine incorporation into nucleoli decreases markedly in both species, whereas the activity of nucleolar RNA polymerase remains at a high level in all root segments inZea mays and decreases slightly inTulipa kaufmanniana.It is argued that the differences between the incorporation of³H uridine and that or³H UMP may be caused by a reduction of the pool of endogenous ribonucleoside triphosphates. Marked activities of DNA polymerase and RNA polymerase in cytoplasm are possibly related to the growth and division of plastids and mitochondria.     

ABam HI family of tobacco highly repeated DNA:

ABamHI family of highly repeated DNA sequences of theNicotiana tabacum nuclear genome, denoted as a HRS60-family, was recently isolated. It comprises about 2% of the tobacco nuclear genome. Monomeric units are 182–184 bp long. Members of the HRS60-family isolated till now are closely related. DNA-DNA hybridization experiments with DNA of the two tobacco progenitors,N. tomentosiformis andN. sylvestris, revealed that the HRS60-family was present in many copies inN. sylvestris, the amount being about 1.7 times that inN. tabacum. InN. tomentosiformis as well as in some other species of the genusNicotiana, the HRS60-family is present in a small amount. Sequences related to the HRS60-family were revealed using DNA-DNA hybridization at low stringency. With respect to quantity, the HRS60-family could be considered as a species-specific DNA repeat which may be a useful genetic marker in genetic manipulations withN. tabacum.     

Optimization of DNA delivery by three classes of hybrid nanoparticle/DNA complexes

Plasmid DNA encoding a luciferase reporter gene was complexed with each of six different hybrid nanoparticles (NPs) synthesized from mixtures of poly (D, L-lactide-co-glycolide acid) (PLGA 50:50) and the cationic lipids DOTAP (1, 2-Dioleoyl-3-Trimethyammonium-Propane) or DC-Chol {3β-[N-(N', N'-Dimethylaminoethane)-carbamyl] Cholesterol}. Particles were 100-400 nm in diameter and the resulting complexes had DNA adsorbed on the surface (out), encapsulated (in), or DNA adsorbed and encapsulated (both). A luciferase reporter assay was used to quantify DNA expression in 293 cells for the uptake of six different NP/DNA complexes. Optimal DNA delivery occurred for 10⁵ cells over a range of 500 ng - 10 μg of NPs containing 20-30 μg DNA per 1 mg of NPs. Uptake of DNA from NP/DNA complexes was found to be 500-600 times as efficient as unbound DNA. Regression analysis was performed and lines were drawn for DNA uptake over a four week interval. NP/DNA complexes with adsorbed NPs (out) showed a large initial uptake followed by a steep slope of DNA decline and large angle of declination; lines from uptake of adsorbed and encapsulated NPs (both) also exhibited a large initial uptake but was followed by a gradual slope of DNA decline and small angle of declination, indicating longer times of luciferase expression in 293 cells. NPs with encapsulated DNA only (in), gave an intermediate activity. The latter two effects were best seen with DOTAP-NPs while the former was best seen with DC-Chol-NPs. These results provide optimal conditions for using different hybrid NP/DNA complexes in vitro and in the future, will be tested in vivo.     

Investigation of liver binding of pentachlorophenol based upon measurement of protein adducts

Abstract

Covalent binding of reactive metabolites of pentachloropheno (PCP) was investigated both in vitro andin vivo in the livers of male Sprague-Dawley rats via measurement of protein adducts. Cysteinyl adducts of quinones andsemiquinones in liver cytosolic (Cp) andnuclear (Np) proteins were assayed after catalytic cleavage by Raney nickel. Results from in vitro experiments confirmed that PCP metabolism produced tetrachlorobenzoquinones andthe comsponding tetrachlorobentosemiquinones which subsequently bound to sulphydryl groups in liver proteins. In vivo, the production of cysteinyl adducts increased with the administered dosage (0–40 mg PCP per kg body weight) andpresented evidence of saturable metabolism. Results suggest two metabolic pathways for PCP, including a high-affinity low-capacity pathway anda low-affinity high-capacity pathway. Time-course experiments in vivo andin vitro suggested that quinone adducts partlcipated in multiple substitution reactions with protein and/or non-protein thiols, andpointed to possible formation of protein-protein cross-links in vivo. The elimination rate constants of quinone adducts in vitro were about 0.35 h^?1 in liver Cp. The elimination of quinone adducts in vivo appeared to follow biphasic kinetics with rate constants for the terminal phase being 0.014 and0.008 h^?1 in liver Cp andNp, respectively.     

The role of the HCR system in the repair of lethal lesions ofBacillus subtilis phages and their transfecting DNA damaged by radiation and alkylating agents

The role of the HCR system in the repair of prelethal lesions induced by UV-light, γ-rays and alkylating agents was studied in theBacillus subtilis SPP1 phage, its thermosensitive mutants (N3, N73 endts ₁) and corresponding infectious DNA. The survival of phages and their transfecting DNA after treatment with UV light is substantially higher inhcr ⁺ cells than inhcr cells, the differences being more striking in intact phages than in their transfecting DNA’s. Repair inhibitors reduce the survival inhcr ⁺ cells: caffeine lowers the survival of UV-irradiated phage SPP1 in exponentially growinghcr ⁺ cells but has no effect on its survival in competenthcr ⁺ cells; acriflavin and ethidium bromide decrease the survival of UV-irradiated SPP1 phage in both exponentially growing and competenthcr ⁺ cells to the level of survival observed inhcr cells; moreover, ethidium bromide lowers the number of infective centres inhcr ⁺ cells of UV-irradiated DNA of the SPP1 phage. Repair inhibitors do not lower the survival of UV-irradiated phages or their DNA inhcr cells. The repair mechanism under study repairs effectively also lesions induced by polyfunctional alkylating agents in transfecting DNA’s ofB. subtilis phages but is not functional with lesions induced by these agents in free phages and lesions caused in phages and their DNA by ethyl methanesulphonate or γ-rays.     

Induction of abnormal male gametes and androgenesis in the aquatic PhycomyceteAllomyces

Summary Induction of cleavage of the cytoplasm in the gametangium of a predominantly male strain of the aquatic PhycomyceteAllomyces under conditions of oxygen starvation, in the presence of dilute lactic acid, or dilute phosphate buffer, pH 7.0, resulted in multinucleate-multi-flagellate gametes. Under certain conditions the frequency for such abnormal gametes approached 70%. Phosphate buffer pH 7.0, at a concentration of 5 × 10^–3-1×10^–2 M and an incubation time of 90 minutes was found to be most effective in inducing multi-flagellate-multinucleate gametes. Nuclear fusion and multiple nuclear fusions were observed in these abnormal gametes as they developed into sporophytic thalli on dilute nutrient agar (YpSS/10). Multinuclear gamete development and nuclear fusion was analysed by electron microscopy. The reported observations reveal the occurrence of androgenesis from multinuclear male gametes inAllomyces.     

DNA Digestion and Chromatin Condensation during Nuclear Death inTetrahymena

DNA fragmentation and nuclear condensation are key features in the regulated cell death of higher animal cells. Nuclear death also occurs as part of a developmentally programmed process during the sexual life cycle of the unicellular organismTetrahymena.We examined the regulation of nuclear death and the relationship between DNA fragmentation and chromatin condensation in this model system. Nuclear death is accompanied by DNA digestion to low-molecular-weight oligonucleosomal-length fragments, in agree- ment with a previous study [17], indicating an endonuclease-like activity typical of apoptosis in higher organisms. Actinomycin D and cycloheximide block DNA digestion as well as nuclear condensation suggesting that nuclear death is under genetic regulation. DNA digestion is completely blocked by aurin, a general nuclease inhibitor. In addition, when DNA fragmentation is blocked, nuclear condensation also fails to occur. Moreover, a kinetic analysis of DNA breakdown, using agarose gels, shows that some DNA digestion occurs before nuclear condensation has taken place. Thus the initiation of DNA digestion may provide conditions necessary for nuclear condensation. Temporary inhibition of nuclear death aborts the death program since after removal of inhibitors cells revert to a vegetative pathway without having eliminated the old or developed the new macronucleus. Zn²⁺and EGTA, both of which inhibit apoptosis in some cell types, fail to prevent nuclear condensation or DNA digestion inTetrahymena,suggesting a requirement here for an endonuclease which is Ca²⁺-independent and Zn²⁺-insensitive. With the TUNEL assay, DNA breakdown is detected exclusively in the condensed macronucleus (and occasional micronuclei identified as degenerating haploid products of meiosis), but not in precondensed macronuclei. These studies show that apoptotic-like DNA fragmentation occurs after condensation of the degenerating macronucleus. However, early DNA digestion may be critical for nuclear condensation and subsequent degeneration.     

湖南南部植物区系新资料

本文报道了湖南双子叶植物1个新记录属、2个新记录种以及1个新记录变种,即中华青牛胆 ［Tinospora sinensis (Loureiro) Merrill］、柳叶润楠(Machilus salicina Hance)、广西白背叶(Mallotus apelta var. kwangsiensis F. P. Metcalf)、赛葵 ［Malvastrum coromandelianum (Linnaeus) Garcke］,赛葵对应的赛葵属(Malvastrum A. Gray)为湖南植物新记录属。入侵植物赛葵可能会对当地的农业生产和生态造成破坏。     

Counterselection of GATC sequences in enterobacteriophages by the components of the methyl-directed mismatch repair system

Summary Weak to severe deficit of GATC sequences in the DNA of enterobacteriophages appears to be correlated with their undermethylation during growth indam ⁺ (GATC ade-methylase) bacteria. This observation is corroborated by the sequence analysis showing no evidence for site-specific mutagenicity of 6meAde. The MutH protein of the methyl-directed mismatch repair system recognizes and cleaves the undermethylated GATC sequences in the course of mismatch repair. To enquire whether the MutH function of the methyldirected mismatch repair system participates in counterselection of GATC sequences in enterobacteriophages, we have studied the yield of bacteriophage X174 containing either 0, 1, or 2 GATC sequences, in wild type,dam, andmut (H, L, S, U) Escherichia coli. Following transfection with unmethylated DNA containing two GATC sequences, a net decrease in the yield of infective particles was observed in all bacterialmutH ⁺ dam^– strains, whereas no detectable decrease was observed in bacteria infected by DNA without GATC sequence. This effect of the MutH function is maximum in wild type andmutL andmutS bacteria whereas the effect is not significant inmutU bacteria, suggesting an interaction of the, helicase II with the MutH protein.However, indam ⁺ bacteria, the presence of GATC sequences leads to an increased yield of infective particles. The effect of GATC sequence and its Dam methylation system on phage yield inmutH ^– bacteria reveals that methylated GATC sequences are advantageous to the phage. These results suggest that the methyl-directed mismatch repair system, and in particular its MutH protein, may have participated in severe counterselection of GATC sequences from enterobacteriophages, presumably, by DNA cleavage or by interfering with DNA replication or packaging when GATC sequences are undermethylated. Coevolution of the Dam and MutH proteins could then account for the loss of GATC sequences from DNA of bacteriophages growing indam ⁺ hosts.     

Rapporti Embrione-Endosperma in Triticum Durum: Velocità Di Utilizzazione,Da Parte Dell'Embrione,Di 3H-Tdr. Proveniente Dall'Endosperma

Abstract

Embryo-endosperm relationship in «Triticum durum» seeds: embryo utilization speed of ³H-Tdr from the endosperm. — The transferring speed of DNA labeled precursor (³H-Tdr) from endosperm to embryo, in Triticum, has been detected by embryo transplantation technique. The results show that the first hour (after transplant) some cells (3%) in root meristem incorporated ³H-Tdr. Labeled cells frequency is increasing between the first and the third hour of experiment, up to 25%; thereafter the percentage keeps constant. The histological location of cells and/or an insufficient availability of ³H-Tdr (from the endosperm) might explain the low labeling index detected in the above experiment.     

Prophage induction in Escherichia coli K12 cells deficient in DNA polymerase I.

Summary The induction of prophage by ultraviolet light has been measured inE. coli K12 lysogenic cells deficient in DNA polymerase I. The efficiency of the induction process was greater inpolA1 polC(dnaE) double mutants incubated at the temperature that blocks DNA replication than inpolA ⁺ polC single mutants. Similarly, thepolA1 mutation sensitizedtif-promoted lysogenic induction in apolA1 tif strain at 42°. In strains bearing thepolA12 mutation, which growth normally at 30°, induction of the prophage occured after the shift to 42°. It is concluded that dissapearance of the DNA polymerase I activity leads to changes in DNA replication that are able, per se, to trigger the prophage induction process.     

Extra DNA synthesis in the dedifferentiating cells ofVicia faba roots

Summary Cell dedifferentiation was induced inVicia faba root tissues by removing the whole root meristem (decapitation) and the behaviour of the nuclear DNA in the dedifferentiating cells was studied by means of cytophotometric and autoradiographic analyses. Cytophotometric determination after Feulgen-staining showed that: 1. the vast majority of nuclei in differentiated cells were in the DNA postsynthetic phase, but their Feulgen absorption was lower than that of DNA postsynthetic nuclei (G₂, 4 C) in the meristem; 2. such a Feulgen absorption was detected in certain nuclei after root decapitation; 3. all the mitoses in the dedifferentiating tissues were diploid, fully matching the Feulgen absorption of mitoses in the meristem.After³H-thymidine (³H-T) feeding of the decapitated roots and autoradiography, the following results were obtained: 1. two populations of labeled nuclei, characterized by two different levels of scattered labeling occurred in dedifferentiating tissues, slightly labeled nuclei being much more numerous than heavily labeled nuclei; 2. the percentage of labeled nuclei was much greater than that of DNA presynthetic nuclei in the root tissues; 3. almost all the mitoses were labeled after a 16-hour³H-T feeding; 4. the percentage of slightly labeled nuclei paralleled that of dedifferentiating cells; 5. the duration of the DNA synthesis phase and that of the gap between completion of DNA synthesis and mitosis differed in heavily and slightly labeled nuclei; 6. all nuclei which entered DNA synthesis also entered mitosis.These results are interpreted to mean that: 1. after decapitation, two different DNA syntheses occur in the dedifferentiating root tissues ofV. faba: DNA reduplication in cells which dedifferentiate starting from a DNA presynthetic nuclear condition (heavily labeled nuclei) and extra DNA synthesis in cells which dedifferentiate starting from a DNA postsynthetic nuclear condition (slightly labeled nuclei); 2. extra DNA synthesis is required in these dedifferentiating cells for entry into mitosis.     

Flow cytometric measurement of nuclear DNA content inCapsicum (Solanaceae)

Nuclei were isolated from leaf tissue of differentCapsicum species and the relative fluorescence intensity was measured by flow cytometry after propidium iodide staining.Pisum sativum nuclei with known nuclear genome size (9.07 pg) were used as internal standard to determine nuclear DNA content of the samples in absolute units. The 2C DNA contents ranged between 7.65 pg inC. annuum and 9.72 pg inC. pubescens, and the general mean of the genus was 8.42 pg. These values correspond, respectively, to 1C genome size of 3.691 (C. annuum), 4.690 (C. pubescens) and 4.063 (general mean) Mbp. In general, white-flowered species proved to have less DNA, with the exception ofC. praetermissum, which displayed a 2C DNA content of 9.23 pg. It was possible to divide the studied species into three main groups according to their DNA content, and demonstrate differences in DNA content within two of the three species complexes established on the basis of morphological traits.     

Expression ofStreptomyces melC andchoA genes by a clonedStreptococcus thermophilus promoter STP2201

Streptococcus thermophilus (ST) chromosomal DNA (chr DNA) fragments having promoter activity were cloned and selected inEscherichia coli using a chloramphenicol acetyltransferase- (cat-) based promoter-probe vector pKK520-3. Insertion of a promoterless streptomycete melanin biosynthesis operon (melC) downstream from the promoters of the library further identified clone ST_P2201 as a strong promoter inE. coli. Subcloning of a ST_P2201-melC DNA fragment into the pMEU-seriesS. thermophilus-E. coli shuttle vectors yielded pEU5xML2201x plasmids that conferred Mel⁺ phenotype toE. coli. The pEU5aML2201a was further shown to afford a high level of tyrosinase production (2 units mg^–1 protein) inE. coli, and to produce an apparently inactivemelC gene product that reacts with anti-tyrosinase antiserum inS. thermophilus. SubstitutingmelC with a streptomycete cholesterol oxidase gene (choA) in the same orientation yielded pEU5aCH2201a that conferred ChoA activity to anE. coli transformant at a level of (1.06±0.15)×10^–7 units mg^–1 protein. Introduction of this plasmid intoS. thermophilus by electrotransformation yielded ChoA transformant that produced the enzyme at about 25% of the level found inE. coli.     

Agar Analysis,nuclear genome quantification and characterization of four agarophytes (Gracilaria) from the Mexican Gulf Coast

DNA reassociation kinetics were used to determine nuclear genome organization and complexity in four species of Gracilaria (Gracilariales, Rhodophyta). In Gracilaria tikvahiae, G. caudata, G. cervicornis and G. divaricata, results indicate the presence of three second order components corresponding to fast, intermediate and slow fractions. Repetitive sequences varied from 13–46% and unique DNA ranged from 45–78%, Thermal denaturation (T _m) indicated guanine + cytosine (G + C) levels of 41.9–46.0 mol % G + C. Microspectrophotometry with the DNA-localizing fluorochrome DAPI was used to quantify nuclear DNA content. Comparisons of mean nuclear DNA (I _f) values to chicken erythrocytes (RBC) resulted in an estimate of 0.37–0.40 pg/2C genomes for the four Gracilaria species. Total agar content following alkaline pretreatment ranged from 7–15% dry weight. Gel strengths were generally below commercial levels, ranging from 40–260 g cm⁻² Nuclear genome profiles developed from information for genome size, organization and complexity are compared with data for agar quantity and quality. Gel quality and quantity do not appear to be correlated with either large repetitive fraction DNA or a high degree of genome complexity as previously speculated.     

Behavior of organelle nuclei (nucleoids) in generative and vegetative cells during maturation of pollen inLilium longiflorum andPelargonium zonale

Summary The behavior of organelle nuclei during maturation of the male gametes ofLilium longiflorum andPelargonium zonale was examined by fluorescence microscopy after staining with 4,6-diamidino-2-phenylindole (DAPI) and Southern hybridization. The organelle nuclei in both generative and vegetative cells inL. longiflorum were preferentially degraded during the maturation of the male gametes. In the mature pollen grains ofL. longiflorum, there were absolutely no organelle nuclei visible in the cytoplasm of the generative cells. In the vegetative cells, almost all the organelle nuclei were degraded. However, in contrast to the situation in generative cells, the last vestiges of organelle nuclei in vegetative cells did not disappear completely. They remained in evidence in the vegetative cells during germination of the pollen tubes. InP. zonale, however, no evidence of degradation of organelle nuclei was ever observed. As a result, a very large number of organelle nuclei remained in the sperm cells during maturation of the pollen grains. When the total DNA isolated from the pollen or pollen tubes was analyzed by Southern hybridization with a probe that contained therbc L gene, for detection of the plastid DNA and a probe that contained thecox I gene, for detection of the mitochondrial DNA, the same results were obtained. Therefore, the maternal inheritance of the organelle genes inL. longiflorum is caused by the degradation of the organelle DNA in the generative cells while the biparental inheritance of the organelle genes inP. zonale is the result of the preservation of the organelle DNA in the generative and sperm cells. To characterize the degradation of the organelle nuclei, nucleolytic activities in mature pollen were analyzed by an in situ assay on an SDS-DNA-gel after electrophoresis. The results revealed that a 40kDa Ca²⁺-dependent nuclease and a 23 kDa Zn²⁺ -dependent nuclease were present specifically among the pollen proteins ofL. longiflorum. By contrast, no nucleolytic activity was detected in a similar analysis of pollen proteins ofP. zonale.     

Correlation between nuclear DNA values and differing optimal ploidy levels inNarcissus,Hyacinthus andTulipa cultivars

4C nuclear DNA amounts were determined in 16 large decorative cultivars ofNarcissus (Amaryllidaceae), 13 ofHyacinthus (Hyacinthaceae) and 12 ofTulipa (Liliaceae) at different levels of ploidy. Within each genus, nuclear DNA amounts and ploidy levels are positively correlated, with no DNA loss in polyploids.Based on wide surveys of chromosome numbers, the maximum numbers of cultivars, interpreted as the optimum levels of selective success or horticultural fitness, were found to be at the tetraploid level inNarcissus (2n=4x=28), the triploid inHyacinthus (2n=3x=24) and the diploid inTulipa (2n=2x=24). All these ploidy optima were shown to correspond to a small range of nuclear DNA amounts (4C=96-139 pg), which could suggest the existence of a single DNA value optimal for the three biologically similar but unrelated genera. In each case the optimum is at an equilibrium reached between enhanced size and other morphological characteristics on one hand and reduced growth rate on the other, both resulting from increase in ploidy and nuclear DNA amounts.