Giemsa C-banded Karyotypes of Eight Species of Alstroemeria L. and Some of Their Hybrids

Karyotype analysis of Alstroemeria angustifolia ssp. angustifolia,A. aurea, A. inodora, A. ligtu spp. ligtu, A. magnifica ssp.magnifica, A. pelegrina, A. philippii and A. psittacina usingFeulgen-staining and Giemsa C-banding techniques revealed foreach species a characteristic chromosome morphology and C-bandingpattern. These characteristics could be used to identify manyindividual chromosomes in diploid interspecific hybrids. Besidesinterspecific variation, some degree of intraspecific variationin C-banding pattern was observed within A. angustifolia ssp.angustifolia, A. aurea, A. ligtu ssp. ligtu, A. magnifica ssp.magnifica and A. philippii . All species had large chromosomes (2 n =2 x =16) and asymmetrickaryotypes. In many species the short arms of the acrocentricchromosomes were darkly stained upon Giemsa C-banding. Thesetelomeric bands seemed satellites. B-chromosomes were observedin one species, A. angustifolia ssp. angustifolia . A variablenumber of large intercalary and telomeric C-bands was presentin the Chilean species, whereas the Brazilian species showedonly small C-bands. The differences in karyotypes suggest anearly separation of the Chilean and Brazilian species, afterwhich speciation followed different evolutionary pathways. InAlstroemeria the Giemsa C-banding technique can be valuableto plant taxonomists for unravelling species relationships. Alstroemeria ; Inca lily; evolution; Giemsa C-banding; karyotype     

Nuclear DNA Amounts in Roses

Nuclei isolated from young leaves were stained with propidiumiodide (PI) and their fluorescence intensities were measuredby flow cytometry. The ratio of fluorescence intensities offour calibration standards and 34 roses to an internal standard,parsley (Petroselinum crispum), provided a basis for estimatingthe DNA amounts of P. crispum and rose. The 2C DNA amount ofP. crispum(2 n = 22) was estimated as 4.46 pg (s.d. ±0.08 pg). The 2C DNA amounts of diploid roses (2n = 14) variedbetween subgenera, sections and cultivars, and ranged from 0.78pg (s.d. ± 0.08 pg) in Rosa xanthina and R. sericea(sectionPimpinellifoliae) to 1.29 pg (s.d. ± 0.08 pg) in ‘Félicitéet Perpétue’ (Hybrid Sempervirens). Within eachsection, the DNA amounts of diploid species were similar. Inthe sections Carolinae and Cinnamomeae, DNA amounts were proportionalto ploidy numbers. In the Pimpinellifoliae, DNA amounts of tetraploidswere disproportionately larger than those of diploids whichsuggests that they originated as hybrids with species of sectionswith larger DNA amounts. Ratios of the fluorescence intensitiesof nuclei of roses to P. crispum(internal standard) were alsomeasured using 4',6-diamidino-2-phenylindole (DAPI) which bindspreferentially to AT base pairs. These DAPI ratios were lowerthan, but closely correlated (r² = 0.997) with PI ratios. Fluorescenceintensities of either PI or DAPI-stained nuclei of roses canbe used as rapid indicators of ploidy if variation in the DNAamounts between different taxonomic groups is taken into account.Copyright 2000 Annals of Botany Company Flow cytometry, nuclear DNA amounts, Petroselinum crispum, phenolics, Rosa, roses     

Genome size variation and C-band polymorphism inAlstroemeria aurea, A. ligtu andA. magnifica (Alstroemeriaceae)

Among a total of 43 accessions ofAlstroemeria aurea, A. ligtu andA. magnifica nuclear DNA amounts (2C-values) showed significant intraspecific variation, 1.09, 1.21 and 1.15 fold, respectively, when determined through flow cytometric measurements of fluorescence of propidium iodide (PI) stained nuclei. After staining with another fluorochrome, 4,6-diamidino-2-phenylindole (DAPI), an intraspecific variation of 1.10, 1.11 and 1.12 fold, respectively, was found. C-band polymorphisms were present among and within the accessions of all three species. In some cases only very small differences in C-banding pattern were observed. In other cases, however, differences were more prominent. Besides C-band polymorphism, there were also instances of chromosome length polymorphism, which concerned the total chromosome complement or single chromosomes. The variation in nuclear DNA amount inA. aurea andA. ligtu was more or less continuous, except for one accession ofA. ligtu subsp.simsii. Artificial selection and possibly introgression of chromosomes from other species may have moulded the karyotypes of some of the accessions ofA. aurea, a species that has been under cultivation for more than 160 years. The variation as observed inA. magnifica subsp.magnifica was discontinuous and could be due to a broad species concept.     

Nuclear DNA Content as an Index Character Discriminating Taxa in the Genus Petunia sensu Jussieu (Solanaceae)

Nuclear DNA contents over the total range of the genus Petuniasensu Jussieu comprising 20 taxa of Petunia sensu Wijsman (2n = 2 x = 14) and 32 taxa of Calibrachoa(2 n = 2 x = 18) wereestimated by flow cytometry after staining the nuclei with propidiumiodide (PI) or 4',6-diamidino-2-phenylindole (DAPI). With respectto nuclear DNA content, taxa of Petunia sensu Wijsman seemedto be homogeneous (2C = 2.60 to 3.41 pg), but Calibrachoa taxawere clearly separated into two groups: (1)C. parviflora andC. pygmaea(1.56 to 1.91 pg); and (2) remaining members of Calibrachoa(2.84to 3.26 pg). Taxa of Petunia sensu Wijsman exhibited largerPI/DAPI ratios (relative fluorescence intensity with PI stainingto that with DAPI staining) than Calibrachoa species exceptC.parviflora and C. pygmaea. This suggests that Petunia sensuWijsman has nuclear DNA with more adenine-thymine rich regionsthan Calibrachoa. Copyright 2000 Annals of Botany Company Calibrachoa, 4',6-diamidino-2-phenylindole (DAPI), flow cytometry, nuclear DNA content,Petunia , PI/DAPI ratio, propidium iodide (PI), Solanaceae     

Characterisation of distant Alstrogmeria hybrids: application of highly repetitive DNA sequences from A. ligtu ssp. ligtu

Clones from a Sau 3A family of eight highly repetitive sequences previously isolated from a genomic DNA library of Alstroemeria ligtu ssp. ligtu were sequenced and found to be highly conserved. A trinucleotide microsatellite repeat [GCA]_3?4 was present. A second, unrelated, Sau 3A repeat was also characterised. Southern analysis proved that the isolated repeats were specific for the A. ligtu subspecies and could not be detected in other Chilean or Brazilean Alstroemeria species. As shown by in situ hybridisation, the Sau 3A family and the unrelated Sau 3A repeat co‐localised at distinct sites along most chromosomes of Alstroemeria ligtu ssp. ligtu and Alstroemeria ligtu ssp. simsii. The present set of species‐specific repetitive sequences enables the identification of A. ligtu chromosomes, and thus the tracking of chromosome transmission to interspecific hybrids and their progeny.     

Nuclear DNA C-values Complete Familial Representation in Gymnosperms

The gymnosperms are a monophyletic yet diverse group of woodytrees with approx. 730 extant species in 17 families. A recentsurvey showed that DNA C-values were available for approx. 16%of species, but for only 12 of the 17 families. This paper completesfamilial representation reporting first C-values for the fiveremaining families: Boweniaceae, Stangeriaceae, Welwitschiaceae,Cephalotaxaceae and Sciadopityaceae. C-values for nine Ephedraand two Gnetum species are also reported. C-values are now availablefor 152 (21%) species. Analysis confirms that gymnosperms arecharacterized by larger C-values than angiosperms (modal 1Cof gymnosperms = 15.8 pg compared with 0.6 pg in angiosperms)although the range (1C = 2.25–32.20 pg) is smaller thanthat in angiosperms (1C = 0.05–127.4 pg). Given completefamilial coverage for C-values and increasing consensus in gymnospermphylogeny, the phylogenetic component of C-value variation wasalso investigated by comparing the two datasets. This analysisrevealed that ancestral gymnosperms (represented by cycads and/orGinkgo; mean genome size = 14.71 pg) probably had larger genomes thanancestral angiosperms. Copyright 2001 Annals of Botany Company Gymnosperm DNA amounts, C-values, phylogeny, ancestral genome size, Cycadales, Ginkgo, Gnetales, conifers, Pinaceae     

Flow cytometric and microscopic analysis of the effect of tannic acid on plant nuclei and estimation of DNA content

• Background and Aims Flow cytometry (FCM) is extensivelyused to estimate DNA ploidy and genome size in plants. In orderto determine nuclear DNA content, nuclei in suspension are stainedby a DNA-specific fluorochrome and fluorescence emission isquantified. Recent studies have shown that cytosolic compoundsmay interfere with binding of fluorochromes to DNA, leadingto flawed data. Tannic acid, a common phenolic compound, maybe responsible for some of the stoichiometric errors, especiallyin woody plants. In this study, the effect of tannic acid onestimation of nuclear DNA content was evaluated in Pisum sativumand Zea mays, which were chosen as model species. • Methods Nuclear suspensions were prepared from P. sativumleaf tissue using four different lysis buffers (Galbraith's,LB01, Otto's and Tris.MgCl₂). The suspensions were treated withtannic acid (TA) at 13 different initial concentrations rangingfrom 0·25 to 3·50 mg mL^–1. After propidiumiodide (PI) staining, samples were analysed using FCM. In additionto the measurement of nuclei fluorescence, light scatter propertieswere assessed. Subsequently, a single TA concentration was chosenfor each buffer and the effect of incubation time was assessed.Similar analyses were performed on liquid suspensions of P.sativum and Z. mays nuclei that were isolated, treated and analysedsimultaneously. FCM analyses were accompanied by microscopicobservations of nuclei suspensions. • Key Results TA affected PI fluorescence and light scatterproperties of plant nuclei, regardless of the isolation bufferused. The least pronounced effects of TA were observed in Tris.MgCl₂buffer. Samples obtained using Galbraith's and LB01 bufferswere the most affected by this compound. A newly described ‘tannicacid effect’ occurred immediately after the addition ofthe compound. With the exception of Otto's buffer, nuclei ofP. sativum and Z. mays were affected differently, with pea nucleiexhibiting a greater decrease in fluorescence intensity. • Conclusions A negative effect of a secondary metabolite,TA, on estimation of nuclear DNA content is described and recommendationsfor minimizing the effect of cytosolic compounds are presented.Alteration in light scattering properties of isolated nucleican be used as an indicator of the presence of TA, which maycause stoichiometric errors in nuclei staining using a DNA intercalator,PI.     

Nuclear DNA Content of 26 Orchid (Orchidaceae) Genera with Emphasis onDendrobium

2C DNA content values for 70 orchid species from 26 genera,including 37Dendrobiumspecies from eight taxonomic sections,were analysed using flow cytometry. The resulting nuclear DNAcontent values for species other thanDendrobiumranged from 1.91pg 2C^-1to 15.19 pg 2C^-1nuclei forCadetia tayloriandVanilla phaeantha,respectively.Dendrobiumnuclear DNA content values ranged from1.53 pg 2C^-1to 4.23 pg 2C^-1nuclei forD. cruentumandD. spectabile,respectively. DNA content measurements varied greatly withinDendrobiumsectionsLatouria and Spatulata. Nuclear DNA content values for the sixspecies analysed within Latouria ranged from 1.88 pg 2C^-1nucleiforD. macrophyllumto 4.23 pg 2C^-1nuclei forD. spectabile. NuclearDNA content values for the 16 species analysed within Spatulataranged from 1.69 pg 2C^-1nuclei forD. discolorto 4.05 pg 2C^-1nucleiforD. samoense. The least variation in DNA content was foundwithin the section Phalaenanthe, with nuclear DNA content valuesof 1.79 pg  2C^-1, 1.86 pg 2C^-1and 1.98 pg 2C^-1forD. bigibbum,D.affineandD. phalaenopsis, respectively.Copyright 1998 Annalsof Botany Company Orchidaceae,Dendrobium, flow cytometry, propidium iodide, nuclear DNA, genome size, 2C values.     

Comparison of four nuclear isolation buffers for plant DNA flow cytometry

• Background and Aims DNA flow cytometry requires preparationof suspensions of intact nuclei, which are stained using a DNA-specificfluorochrome prior to analysis. Various buffer formulas weredeveloped to preserve nuclear integrity, protect DNA from degradationand facilitate its stoichiometric staining. Although nuclearisolation buffers differ considerably in chemical composition,no systematic comparison of their performance has been madeuntil now. This knowledge is required to select the appropriatebuffer for a given species and tissue. • Methods Four common lysis buffers (Galbraith's, LB01,Otto's and Tris.MgCl₂) were used to prepare samples from leaftissues of seven plant species (Sedum burrito, Oxalis pes-caprae,Lycopersicon esculentum, Celtis australis, Pisum sativum, Festucarothmaleri and Vicia faba). The species were selected to covera wide range of genome sizes (1·30–26·90pg per 2C DNA) and a variety of leaf tissue types. The followingparameters were assessed: forward (FS) and side (SS) light scatters,fluorescence of propidium iodide-stained nuclei, coefficientof variation of DNA peaks, presence of debris background andthe number of nuclei released from sample tissue. The experimentswere performed independently by two operators and repeated onthree different days. • Key Results Clear differences among buffers were observed.With the exception of O. pes-caprae, any buffer provided acceptableresults for all species. LB01 and Otto's were generally thebest buffers, with Otto's buffer providing better results inspecies with low DNA content. Galbraith's buffer led to satisfactoryresults and Tris.MgCl₂ was generally the worst, although ityielded the best histograms in C. australis. A combined analysisof FS and SS provided a ‘fingerprint’ for each buffer.The variation between days was more significant than the variationbetween operators. • Conclusions Each lysis buffer tested responded to a specificproblem differently and none of the buffers worked best withall species. These results expand our knowledge on nuclear isolationbuffers and will facilitate selection of the most appropriatebuffer depending on species, tissue type and the presence ofcytosolic compounds interfering with DNA staining.     

Reference standards for determination of DNA content of plant nuclei

Flow cytometry was used to compare 14 potential reference standards for plant DNA content determination. Both chicken and plant internal standards were used, as were propidium iodide (PI) and 4'-6-diamidino-2-phenylindole (DAPI) as fluorochromes. Means and standard errors of the means are presented for the 14 potential reference standards, and the means are compared to those obtained by Feulgen densitometry. Five species are recommended as an initial set of international standards for future plant DNA content determinations: Sorghum bicolor cv. Pioneer 8695 (2C = 1.74 pg), Pisum sativum cv. Minerva Maple (2C = 9.56 pg), Hordeum vulgare cv. Sultan (2C = 11.12 pg), Vicia faba (2C = 26.66 pg), and Allium cepa cv. Ailsa Craig (2C = 33.55 pg). It is recommended that the reference standard of choice be one with 2C and 4C nuclear DNA content peaks similar to, but not overlapping, the 2C and 4C peaks of the target species. We recommend PI as the fluorochrome of choice for flow cytometric determination of plant DNA content. DAPI should be used only if the estimated DNA value is corroborated by using a second stain that has no bias for AT- or GC-rich sequences within genomes.     

DNA Levels in Dividing and Developing Plastids in Expanding Primary Leaves of Avena sativa

The amounts of plastid DNA in the primary leaves of 4-d-oldlight- and dark-grown seedlings of Avena sativa were measuredby microspectrofluorometry using the DNA-fluorochrome DAPI (4',6-diamidino-2-phenylindole). In the light-grown primary leaves (40–45 mm long) therewas a marked increase in DNA level per plastid from 10.2 to18.5 ? 10^–15 g between 2.0 mm and 10 mm from the leafbase, resulting from the rate of plastid DNA synthesis beinghigher than the rate of plastid division. Beyond 30 mm the plastidDNA level was reduced to 14 ? 10^–15g due to chloroplastdivision rates being higher than the rate of plastid DNA synthesis,while from 20 mm plastid DNA levels were constant at 2.2 ? 10^–12g per cell, which corresponds to 16000 plastome copies per cell. Observations of dark-grown leaves establish that, in Avena,light is not necessary for plastid division and the dark-grownleaf cells accumulate higher amounts of plastid DNA than light-grownleaf cells. Plastid nucleoids showed a change of distribution after completionof plastid DNA synthesis in light-grown leaves. A change inthe distribution of plastid nucleoids was also observed duringthe greening of etioplasts of dark-grown leaves while plastidDNA level remained constant. Such changes in plastid nucleoiddistribution appear to be independent of plastid DNA synthesisand correlate with the formation of grana stacks. Key words: Avena sativa, microspectrofluorometry, plastid DNA     

Reverse fluorescent chromosome banding with chromomycin and DAPI

Two DNA binding guanine-specific antibiotics, chromomycin A₃ (CMA) and the closely related mithramycin (MM), were used as chromosome fluorescent dyes. Root-tip metaphase chromosomes of three plant species and human metaphase chromosomes were sequentially stained with CMA or MM and the DNA binding AT-specific fluorochrome 4-6-diamidino-2-phenylindole (DAPI). In some cases a non-fluorescent counterstain was used as contrasting agent: methyl green in conjunction with CMA, and actinomycin D (AMD) in combination with DAPI. — In all three plant species, Vicia faba, Scilla siberica, and Ornithogalum caudatum, the nucleolus organiser regions and/or associated heterochromatin displayed very bright fluorescence with CMA and MM and, in general, heterochromatic segments (C-bands) which were bright with CMA and MM were pale with DAPI whereas segments which were dim with CMA and MM displayed very bright fluorescence with DAPI. — Human metaphase chromosomes showed a small longitudinal differentiation in CMA fluorescence, which was essentially the reverse of the banding pattern obtained with AMD/DAPI double-staining, but of lower contrast. The CMA-banding pattern appears to be similar to the pattern found by R-banding procedures.     

Sunflower (Helianthus annuus) Leaves Contain Compounds that Reduce Nuclear Propidium Iodide Fluorescence

Sunflower leaves have unidentified compounds that interferewith propidium iodide (PI) intercalation and/or fluorescence.Independently prepared pea leaf nuclei show greater PI fluorescencethan nuclei from pea leaves simultaneously processed (co-chopped)with sunflower leaves. Differences in fluorescence persist aftermixing the PI-stained pea and the co-chopped pea/sunflower samples,i.e. PI staining protects the nuclei from the effects of theinhibitor. The current results are significant to practicalflow cytometric determination of plant nuclear DNA content.They show: (1) simultaneous processing of nuclear samples fromthe target and the standard species is necessary to obtain reliableDNA estimates; (2) a test for the presence of inhibitors shouldbe conducted; and (3) when inhibitors are present caution shouldbe taken in interpreting differences in estimated DNA content.The previously reported environmentally-induced variation inDNA content in sunflower populations is most simply explainedby variation in the amount of environmentally-induced inhibitorthat interferes with intercalation and/or fluorescence of PI.Intraspecific variation of DNA content for Helianthus annuusneeds to be re-evaluated using best practice techniques comparingphysiologically uniform tissues that are free of inhibitors.The best estimate for 2C DNA content of H. annuus used in thisstudy is 7.3 pg. Copyright 2000 Annals of Botany Company Helianthus annuus, DNA content, flow cytometry, propidium iodide, endogenous inhibitors     

Photosynthetic Characteristics of C3-C4 Intermediate Flaveria Species II. Kinetic Properties of Phosphoenolpyruvate Carboxylase from C3, C4 and C3-C4 Intermediate Species

The kinetic properties of phosphoenolpyruvate (PEP) carboxylasehave been studied among several Flaveria species: the C₃ speciesF. cronquistii, the C₃–C₄ species F. pubescens and F.linearis, and the C₄ species F. trinervia. At either pH 7 or8, the maximum activities (in µmol.mg Chl^–1.h^–1)for F. pubescens and linearis (187–513) were intermediateto those of the C₃ species (12–19) and the C₄ species(2,182–2,627). The response curves of velocity versusPEP concentration were hyperbolic for the C₃ and C₃–C₄species at either pH 7 or 8 while they were sigmoidal for theC₄ species at pH 7 and hyperbolic at pH 8. The K_m values forPEP determined from reciprocal plots were lowest in the C₃ species,and of intermediate value in the C₃–C₄ species comparedto the K' values of the C₄ species determined from Hill plotsat either pH 7 or 8. Glucose-6-phosphate (G6P) decreased theK_m values for PEP at both pH 7 and 8 in the C₃ and C₃–C₄species. In the C₄ species, G6P decreased the K' values at pH8 but increased the K' values at pH 7. In all cases, G6P hadits effect by influencing the activity at limiting PEP concentrationswith little or no effect on the maximum activity. At pH 8 andlimiting concentrations of PEP the degree of stimulation ofthe activity by G6P was greatest in the C₄ species, intermediatein F. linearis, a C₃–C₄ species, and lowest in the C₃species. In several respects, the PEP carboxylases of the C₃–C₄Flaveria species have properties intermediate to those of theC₃ and C₄ species. (Received April 30, 1983; Accepted August 22, 1983)     

Characterization of geographically isolated accessions in five Alstroemeria L. species (Chile) using FISH of tandemly repeated DNA sequences and RAPD analysis

In fifteen geographically isolated populations of five species of Alstroemeria L. (A. aurea, A. hookeri, A. ligtu, A. pelegrina and A. presliana) collected in Chile, karyotypes and variation of RAPD markers were investigated. Tandemly repeated DNA sequences - 5S and 18/25S rDNA genes and the sequence A001-1 (De Jeu et al. 1997) were used to characterize karyotypes by fluorescence in situ hybridization (FISH). Ten somatic metaphases per population were used for measurement of chromosome length. Differences in RAPD marker bands were used for characterization of populations, creating a similarity index. FISH with all three DNA probes shows a high degree of polymorphism between and sometimes also within accessions of A. aurea, A. hookeri and A. ligtu. The number of chromosome pairs showing 5S rDNA signals is more different for the investigated species A. aurea, A. hookeri, A. ligtu, A. pelegrina and A. presliana with 5, 7, 5, 3 and 7, respectively, than the number of 18/25S rDNA signals in this succession with 7, 7, 6, 5 and 7 chromosome pairs, showing a high evolutionary dynamics within the genus. Furthermore, among the four populations of A. hookeri, accession 4181 was different in arm length of chromosome 3. RAPD markers (index of similarity) also showed a greater genetic distance of accession 4181 from the other three accessions of A. hookeri. The possible evolutionary mechanisms providing these polymorphisms were discussed.     

Substantial Genome Size Variation in Taraxacum stenocephalum (Asteraceae, Lactuceae)

There are only a few exceptions to the rule that polyploidy in Taraxacum is associated with agamospermy. One of them is the sexual, tetraploid species Taraxacum stenocephalum. Incidentally, remarkable variation in karyology was found in this species. The present study aims to confirm this variation by an extensive screen of nuclear DNA content. Individuals from two large populations in the Lesser and Greater Caucasus, Georgia were analyzed using flow cytometry to ascertain intraspecific nuclear DNA content variation. Across the whole data set comprising all 159 individuals, a 1.223-fold difference was detected based on propidium iodide (PI) analyses. To verify this finding, we compared flow-cytometric data obtained using DAPI (4′,6-diamidino-2-phenylindole) and PI staining using a representative subset of individuals. This comparison revealed a 1.194-fold difference in DNA content for DAPI and a 1.219-fold difference for PI. Mean nuclear genome size in absolute terms (2C value ± s.d.) was estimated at 4.38?±?0.21 pg, ranging from 4.01 pg to 4.89 pg, despite the invariable chromosome counts of 2n?=?32. A regression analysis comparing the datasets for DAPI and PI staining found a strong correlation between data obtained by the DAPI and PI dyes (R?=?0.976; P?=?0.0001). Simultaneous high-resolution flow-cytometric analyses also proved the accuracy of our findings. We discuss possible sources of these large differences in DNA content within Taraxacum stenocephalum. Further research is needed to identify the source of this remarkable variation.     

The organization of DNA in the mitotic and polytene chromosomes of Sciara coprophila

The organization of DNA in the mitotic metaphase and polytene chromosomes of the fungus gnat, Sciara coprophila, has been studied using base-specific DNA ligands, including anti-nucleoside antibodies. The DNA of metaphase and polytene chromosomes reacts with AT-specific probes (quinacrine, DAPI, Hoechst 33258 and anti-adenosine) and to a somewhat lesser extent with GC-specific probes (mithramycin, chromomycin A₃ and anticytidine). In virtually every band of the polytene chromosomes chromomycin A₃ fluorescence is almost totally quenched by counterstaining with the AT-specific ligand methyl green. This indicates that GC base pairs in most bands are closely interspersed with AT base pairs. The only exceptions are band IV-8A3 and the nucleolus organizer on the X. In contrast, quinacrine and DAPI fluorescence in every band is only slightly quenched by counterstaining with the GC-specific ligand actinomycin D. Thus, each band contains a moderate proportion of AT-rich DNA sequences with few interspersed GC base pairs. — The C-bands in mitotic and polytene chromosomes can be visualized by Giemsa staining after differential extraction of DNA and those in polytene chromosomes by the use of base-specific fluorochromes or antibodies without prior extraction of DNA. C-bands are located in the centromeric region of every chromosome, and the telomeric region of some. The C-bands in the polytene chromosomes contain AT-rich DNA sequences without closely interspered GC base pairs and lack relatively GC-rich sequences. However, one C-band in the centromeric region of chromosome IV contains relatively GC-rich sequences with closely interspersed AT base pairs. — C-bands make up less than 1% of polytene chromosomes compared to nearly 20% of mitotic metaphase chromosomes. The C-bands in polytene chromosomes are detectable with AT-specific or GC-specific probes while those in metaphase chromosomes are not. Thus, during polytenization there is selective replication of highly AT-rich and relatively GC-rich sequences and underreplication of the remainder of the DNA sequences in the constitutive heterochromatin.     

A comparative study of the size-dependent organic composition of marine diatoms and dinoflagellates

Cellular chlorophyll a, protein, carbohydrate and lipid contentwas determined for eleven clones of centric marine diatoms (volume89–1.47 x 10⁷ µ³) and eight species of marine dinoflagellates(597–4.45 x 10⁴ µ³) cultured under continuous illuminationat 18°C and 20°C, respectively. In both groups the logof cellular concentrations of each constituent was directlyrelated to the log of cell volume; diatoms generally had lowercellular concentrations than dinoflagellates of an equivalentvolume. Diatom chlorophyll a, protein and lipid concentrationsnormalized to a unit cell volume (pg µ^–3) decreasedexponentially with increasing cell size; this decrease is aconsequence of the diatoms' unique morphology restricting cellcytoplasm to a thin parietal layer within the frustule. Althoughdinoflagellates yield a wide range of cytoplasm concentrations,small dinoflagellates contained up to 3-fold higher cytoplasmconcentrations of all constituents than diatoms of equal volume.The log of cellular caloric values, summed from the caloricequivalents of cellular protein, carbohydrate and lipid, wasa linear function of log volume. Diatoms contained ca. halfthe caloric value of dinoflagellates of an equivalent volume.Although the evaluation of caloric content provides a basisfor comparing the "nutritional value" of phytoplankton groups,evidence from the literature suggests subjective factors suchas taste and digestibility are equally important in determiningnutritional values of individual species.     

Characterization of regulatory mechanisms and states of human organic cation transporter 2

Polyspecific organic cation transporters (OCTs) have a large substrate binding pocket with different interaction domains. To determine whether OCT regulation is substrate specific, suitable fluorescent organic cations were selected by comparing their uptake in wild-type (WT) human embryonic kidney (HEK)-293 cells and in HEK-293 cells stably transfected with hOCT2. N-amidino-3,5-diamino-6-chloropyrazine-carboxamide (amiloride) and 4-[4-(dimethylamino)-styryl]-N-methylpyridinium (ASP) showed concentration-dependent uptake in hOCT2 at 37°C. After subtraction of unspecific uptake determined in WT at 37°C or in hOCT2 at 8°C saturable specific uptake of both substrates was measured. K_m values of hOCT2-mediated uptake of 95 µM amiloride and 24 µM ASP were calculated. Inhibition of amiloride and ASP uptake by several organic cations was also measured [IC₅₀ (in µM) for amiloride and ASP, respectively, tetraethylammonium (TEA) 98 and 30, cimetidine 14 and 26, and tetrapentylammonium (TPA) 7 and 2]. Amiloride and ASP uptake were significantly reduced by inhibition of Ca²⁺/CaM complex (–55 ± 5%, n = 10 and –63 ± 2%, n = 15, for amiloride and ASP, respectively) and stimulation of PKC (–54 ± 5%, n = 14, and –31 ± 6%, n = 26) and PKA (–16 ± 5%, n = 16, and –18 ± 4%, n = 40), and they were increased by inhibition of phosphatidylinositol 3-kinase (+28 ± 6%, n = 8, and +55 ± 17%, n = 16). Inhibition of Ca²⁺/CaM complex resulted in a significant decrease of V_max (160–99 photons/s) that can be explained in part by a reduction of the membrane-associated hOCT2 (–22 ± 6%, n = 9) as determined using FACScan flow cytometry. The data indicate that saturable transport by hOCT2 can be measured by the fluorescent substrates amiloride and ASP and that transport activity for both substrates is regulated similarly. Inhibition of the Ca²⁺/CaM complex causes changes in transport capacity via hOCT2 trafficking. organic cation transport; fluorescence measurement; 4-[4-(dimethylamino)-styryl]-n-methylpyridinium; amiloride     

First Nuclear DNA C-values for Another 25 Angiosperm Families

Nuclear DNA C-value is an important genomic biodiversity characterwith many uses. An international workshop sponsored by Annalsof Botany and held at the Royal Botanic Gardens, Kew, UK, in1997 identified major gaps in our knowledge of plant DNA C-valuesand recommended targets for new work. Improved taxonomic coveragewas highlighted as a key need for angiosperms, especially atthe familial level. In 1997 C-values were known for only approx.32% of angiosperm families; a goal of complete familial representationby 2002 was recommended. A review published in 2000 (Bennettet al.;Annals of Botany86: 859–909) noted poor progresstowards this aim: of the 691 first C-values for species only12 (1.7%) were for unrepresented families. We began new workto address this in 1999, reporting first DNA C-values for 25angiosperm families in 2001 (Hanson et al.;Annals of Botany87:251–258). Here we report first DNA C-values for a further25 angiosperm families, increasing familial coverage in angiospermsto approx. 45%. Such targeting remains essential to approachthe goal set by the 1997 workshop of familial coverage for angiospermswithin 5 years. The 4C DNA amounts presented here range from0.76 pg (similar toArabidopsis thaliana ) in Roridula gorgonias(Roridulaceae)to 29.74 pg in Gunnera manicata(Gunneraceae). 1C values were< 3.5 pg in 23 of the 25 families; these data provide furthersupport for the view that ancestral angiosperms almost certainlyhad small genomes (defined as 1C