Determining relative abundance of specific DNA sequences in flow cytometrically sorted maize nuclei

A wide breadth of DNA content variation has been reported amongmaize lines. While the extent of this variation has been welldocumented, few studies have focused on its cause. Some of thenuclear DNA content variation has been explained by the presenceof B chromosomes or knobs. However, variation in these two structuresdoes not account for all of the observed variation. In orderto identify other fluctuating DNA sequences, a rapid and reliablemethod of estimating relative abundance of DNA sequences neededto be developed. The potential of flow cytometry in conjunctionwith spot hybridization for determining relative abundance ofspecific DNA sequences in maize was studied. Different numbersof G1 phase nuclei were sorted on nitrocellulose filters andnon-radioactive hybridization and signal detection performed.Results from these experiments revealed a significant, positivelinear correlation between the amount of target sequence andsignal density using both knob (R = 0.98) and ribosomal spacer(R =0.99) DNA sequences. In addition, G1 phase nuclei of eightinbred lines differing in the amount of knob heterochromatin,were sorted on to filters and the non-radioactive hybridizationand signal detection performed. A significant, positive linearcorrelation between C-band number and signal density (R =0.83;P = 0.0051) as well as between per cent heterochromatin andsignal density (R=0.96;P = 0.0002) was observed. These resultsindicate the usefulness of flow cytometry for spot hybridizationin determining the relative abundance of DNA sequences in themaize genome. Key words: Flow cytometry, copy number, non-isotope labelling, spot hybridization, flow sorting, Zea mays L.     

Genome multiplication in cardiomyocytes of fast- and slow-growing mice

The number of myocytes and the percentage of cells with a high degree of ploidy increased in the heart ventricles of fast-growing mice compared with slow-growing ones. The mean incidence of octa- and hexadecaploid (by summary DNA content) myocytes was 7% in the slow-growing and 23% in the fast-growing, weaned mice. In these groups, the total myocyte number varied by 20%. There were 43% more myocyte genomes in the heart ventricles of the fast-growing mice than in those of the slow-growing mice. The same differences in cell number and ploidy persist in 90-day-old mice in spite of feeding ad libitum after weaning.     

Detection of the IAA Biosynthetic Pathway from Tryptophan via Indole-3-Acetamide in Bradyrhizobium spp.

The IAA biosynthetic pathway of tryptophan to IAA via IAM wasdetected in Bradyrhizobium spp. (slow-growing Rhizobium) butnot in Rhizobium spp. (fast-growing Rhizobium). A simple methodusing rapid HPLC analysis to measure the conversion from NAMto NAA was developed to detect indole-3-acetamide hydrolaseactivity in cultures of bacteria. Most of the Bradyrhizobiumstrains produce large amounts of NAA converted from NAM underour assay conditions. In addition, GC/MS analysis of purifiedextracts from cultures of B.japonicum wild-type strain J1063,grown in a tryptophan-supplemented liquid medium, demonstratedthe presence of IAM and IAA. The results strongly suggest thatbiosynthesis of IAA in Bradyrhizobium spp. involves the samepathway as that operating in Pseudomonas savastanoi and Agrobacteriumtumefaciens. (Received December 25, 1988; Accepted May 18, 1988)     

Lack of proportionality between gene dosage and total muscle protein content in the rat heart.

Counting of isolated cardiomyocytes has demonstrated that their number was 16.8 +/- 0.6 10(6) in both ventricles of weanling rats (28 days after birth), growing in litters of four (fast-growing). In rats growing in litters of 16 (slow-growing), the myocyte number was 11.8 +/- 0.8 10(6). In the control group (8 sucklings per litter), there were 14.2 +/- 10(6) cardiomyocytes. The fast-growing rats had more octoploid cells than slow-growing ones. Considering ploidy and cell number, the total number of myocyte genomes in fast-growing animals was 45% higher than in slow-growing ones. The total content of contractile proteins in fast-growing weanling animals was higher by 28% while sarcoplasmic proteins were 8% higher. This lack of correspondence between the number of myocyte genomes and muscle protein content was even more pronounced at the age of 110 days. The results are compared with the cytophotometric data concerning the lack of correspondence between the total protein content in a myocyte and its DNA amount and chromosome number, i.e., total dosage of the myocyte genes.     

Growth Parameters of Yield of Plantain (Musa cv. AAB)

The performance of the crop plant and the ‘ratoon’plant (lateral shoot succeeding the harvested main shoot) of65 medium-sized False Horn plantains (Musa cv. AAB) was studiedby measuring vegetative and inflorescence (bunch) parameters.From significant regressions between different parameters itappears that taller pseudostems produce leaves at a faster rate,flower earlier and produce heavier infructescences which needmore time to mature. A high yield is determined by vigorousinitial growth of the planted sucker (lateral root). Beforeflowering, there is no sink competition between ratoon and mainpseudostem growth, and fast-growing main pseudostems are accompaniedby fast-growing ratoons. After flowering, competition occursbetween the fructifying inflorescence, a preferential sink andthe ratoon. The results indicate that plantain should not beconsidered as an annual crop. Growth, development, production parameters, bananas, plantains     

Karyotype, DNA Content and Meiotic Behaviour in Five South American Species ofVicia(Fabaceae)

This paper presents the karyotype, DNA content and meiotic behaviourof five species ofViciafrom Argentina (V. macrogramineaBurk.,V.gramineaSM.,V. epetiolarisBurk.,V. pampicolaBurk. andV. nanaVog.).All the species have the same chromosome number and karyotypeformula (2n=14; 6m+4st+4t). Each species, however, displaysa characteristic number and position of the nucleolar organizerregion (NOR) and different sizes of the respective satellites,confirmed by Ag-NOR banding. Moreover, significant differenceswere found in the total chromosome volume (TCV) and DNA contentof the species. Positive correlations between DNA content andTCV, and between DNA content and type of life cycle were alsofound. TCV and DNA content are lower inV. nana(annual) and higherinV. macrograminea(biennial–perennial). The material displayedmarked karyotypic orthoselection, with similar karyotypes inall studied species, even when the overall chromosome size varied.Evolutionary changes in DNA amount are proportional to the relativelength of each chromosome arm, maintaining karyotypic uniformity.Significant differences were found between the meiotic behaviourofV. gramineaand that of the other species.V. gramineahas alower frequency of ring bivalents and chiasmata per cell, andalso has a lower interstitial chiasma frequency. In general,the results are congruent with the morphological data reportedfor these species.Copyright 1998 Annals of Botany Company. Viciaspecies, karyotype, orthoselection, nuclear DNA content, NOR banding, meiotic behaviour.     

Variation of Nuclear DNA Content in the Biflorus Species of Lonchocarpus (Leguminosae)

This represents the first study of nuclear DNA content in alarge sample (135 spp.) from a tropical arboreal genus, in whicha large proportion of the species were examined (42 spp., 31.1%).Somatic chromosome numbers and 4C-DNA values for 51 taxa ofLonchocarpus are reported. All taxa were diploid with 2 n =22,but their DNA content ranged from 1.92 to 2.86 pg 4C nucleus,corresponding to a 48.95% variation in genome size. In the 74collections studied, no correlation was observed between DNAcontent and habitat altitude. Variation in nuclear DNA contentwas analysed at the level of genus, subgenus, section and subsection.Variation in genome size was also studied within some species,either among widely separated populations or among differentintraspecific taxa. Very little variation in genome size wasdetected between populations, subspecies, and varieties of thesame species. The taxonomic implications of variation in nuclearDNA content are discussed.Copyright 2000 Annals of Botany Company Lonchocarpus (Leguminosae), DNA content, chromosome number.     

Physical Characterization of the Chromosomal DNA of the Yeast Saccharomyces exiguus

The number of chromosomes in the yeast Saccharomyces exiguuswas determined to be thirteen by two-dimensional pulsed-fieldgel electrophoresis. The thirteen chromosomes ranged in DNAsize from 520 to 2,600 kbp, with a total length of approximately14 Mbp. Numbers I to XIII were assigned to the chromosomes indecreasing order of DNA length. Southern hybridization analysisusing total DNAs from S. exiguus and S. cerevisiae as probesshowed that there was no significant homology between the chromosomalDNAs of the two species, except in the case of the chromosomalDNA that included rDNA. When rDNA and genes LEU2, TRP1, URA3and HO of S. cerevisiae were used as hybridization probes, itwas apparent that S. exiguus had DNA sequences homologous tothe rDNA and to the LEU2 and HO genes. In S. exiguus, rDNA-likeand LEU2-like DNAs were located on chromosomes I and IX, respectively,and HO-like DNA was located on chromosome VI or VII. (Received May 17, 1993; Accepted July 15, 1993)     

Effectiveness of Lotus Root Nodules: II. RELATIONSHIP BETWEEN ROOT NODULE EFFECTIVENESS AND 'IN VITRO' SENSITIVITY OF FAST-GROWING LOTUS RHIZOBIA TO FLAVOLANS

An extract from the roots of Lotus pedunculatus plants was foundto contain a compound toxic towards fast-growing Lotus rhizobia.This compound was identified as a flavolan, which has a prodeiphinidin:procyanidin ratio of 75:25. A fast-growing strain of Rhizobium(NZP2213) which forms ineffective root nodules on L. pedunculatuswas four times more sensitive to this flavolan (ED₅₀ = 25 ?gml^–1) than another strain (NZP2037, ED₅₀ = 100 ?g ml^–1)which forms effective root nodules on this species. The rootsof another Lotus species, L. tenuis, on which both strains ofRhizobium form effective root nodules, also contained a flavolan( 95% procyanidin) but both strains were relatively insensitiveto this flavolan (EDED₅₀ = 350 to 500 ?g ml^–1) L. pedunculatusplants bearing ineffective root nodules contained two to threetimes more flavolan in their roots (5–7 mg g^–1 fr.wt.)than uninoculated control plants. Experiments with seven otherLotus species and with hybrid plants developed between L. pedunculatusand L. tenuis showed a relationship between the prodeiphinidin:procyanidin ratio of the flavolan in their roots and the effectivenessof root nodules formed on these plants by NZP2213. Quantitativebinding studies of the flavolan from L. pedunculatus to NZP2037and NZP2213 indicated that, while the affinity constants forbinding were similar for both strains, the surface of strainNZP2037 contained four times more binding sites than NZP2213,possibly correlating with this strain's ability to toleratehigher concentrations of this flavolan. It is suggested thatthe differential sensitivity of these two strains of Rhizobiumto flavolans is related to their ability to form effective rootnodules on Lotus species.     

Highly Repetitive Tandem Arrays of a Short 32 bp Unit in the Pharbitis nil Genome: the RsaI Family

A highly repetitive DNA sequence of Pharbitis nil, designatedthe RsaI family, was cloned, sequenced and analyzed with respectto its genomic organization. The RsaI family is arranged intandem arrays and composed of a 32 bp repeat unit, which isthe shortest unit thus far reported for plant repetitive sequences.The RsaI family represents 3% of the total genomic DNA and thecopy number of the 32 bp unit is estimated to be about 1 ? 10⁶per haploid genome. We suggest the existence of a higher orderrepeat unit, which may be composed of hundreds of the 32 bpunits and be repeated many times in the genome. (Received May 12, 1988; Accepted July 28, 1988)     

Flow Cytometric Determination of Relative Nuclear DNA Contents in Bicellulate and Tricellulate Pollen

Nuclear DNA content in mature pollen was measured with a flowcytometer Pollen of Lilium longiflorum, Dendranthema grandiflora(syn Chrysanthemum monfolium) and Zea mays was chopped and stainedwith the DNA fluorochrome DAPI DNA levels, expressed as arbitraryC values, were compared with those of nuclei isolated from leafor root material of the same plants In mature tricellulate pollen the generative cell is dividedafter second pollen mitosis into two sperm cells Tricellulatepollen from maize and chrysanthemum gave rise to one large 1Cpeak and, only in the case of chrysanthemum, a much smallerone at the 2C level These results suggest that the haploid nucleiof the vegetative as well as both sperm cells in tricellulatepollen are arrested in the G₁ stage of nuclear division Thesmall 2C peak in the case of chrysanthemum probably arose froma fraction of pollen with the sporophytic chromosome number(2n pollen) In contrast to this, mature bicellulate lily pollengave rise to two identical peaks at the 1C and the 2C levelFrom this result it was concluded that in bicellulate pollen,the 1C peak is caused by the signal of the haploid vegetativenucleus arrested in the G₁ stage of nuclear division, whereasthe 2C peak originates from the haploid generative nucleus whichhas already undergone DNA synthesis and is arrested in G₂ Lilium longiflorumThunb, lily, Dendranthema grandiflora Tzelev (syn Chrysanthemum morifolium Ramat ), chrysanthemum, Zea maysL, maize, male gametophytic cells, vegetative cells, generative cells, sperm cells, unreduced pollen, sporophytic cells, relative nuclear DNA contents, replication stage     

Investigations of Genome Relationships Between Leymus, Psathyrostachys and Hordeum Inferred by Genomic DNA:DNA in situ Hybridization

Genome relationships between the genera Leymus Hochst., PsathyrostachysNevski and Hordeum L. (Poaceae, Triticeae) were investigatedby fluorescent in situ hybridization using both total genomicDNA and cloned DNA sequences as probes. In hybrids between speciesof Hordeum and Leymus there was a clear differentiation betweenthe H genomes of Hordeum species and the genomes of Leymus speciesafter probing with genomic Hordeum or Leymus DNA. Chromosomesof species of Leymus and Psathyrostachys were also differentiatedby subtelomeric heterochromatic segments or by negative bandsalong their length. The number and location of 18S-5·8S-26SrRNA genes varied between the investigated genera. Unusually,L. angustus and P. stoloniformis rDNA sites were localized onboth ends of some chromosomes. Interphase nuclei of the Hordeumx Leymus hybrids had groups of chromosomes from both parentalgenomes in discrete, non-intermixed domains.Copyright 1994,1999 Academic Press Taxonomy, evolution, molecular evolution, repetitive DNA, rDNA sites, in situ hybridization, Triticeae, Leymus, Hordeum, Psathyrostachys     

Molecular Cytogenetics of the Genus Arabidopsis: In situ Localization of rDNA Sites, Chromosome Numbers and Diversity in Centromeric Heterochromatin

We have used in situ hybridization to determine the number ofsites of rDNA in species in the genus Arabidopsis. A. wallichii(2n = 16) has one major pair of sites and one minor pair ofsites, while A. pumila and A. griffithiana (both 2n = 32) havesix major and two minor rDNA sites. A. thaliana (2n = 10) isknown to have two pairs of rDNA sites. a highly repeated para-centromericsequence from A. thaliana, pAL1, is absent in the other threespecies. Hence the A.thaliana genome is not present (or thecentromeric DNA has evolved substantially) in the polyploidspecies A. pumila and A. griffithiana. Analysis of Arabidopsisspecies is a valuable complement to the large programmes forgenetic analysis of A. thaliana.Copyright 1993, 1999 AcademicPress Arabidopsis, centromeric DNA, maps (genetic), nuclear architecture, repetitive DNA, ribosomal DNA, rDNA, evolution, Brassicaceae, Crucifereae, in situ hybridization     

Functional and Structural Chromosome Analyses in Autotetraploid Silene latifolia

Recent immunocytological and molecular data show that heterochromaticnuclear regions, both constitutive and facultative, are modifieddifferently (cytosine hypermethylation and histone hypoacetylation)and late replicating, when compared to euchromatin. Intrusiveand/or additive (supernumerary) DNA sequences are often functionallysilenced; this is accompanied by their heterochromatinization.In this work we present a number of karyological studies onautotetraploid female cells of Silene latifolia (syn. Melandriumalbum). Immunofluorescence analyses do not indicate any globaldifferences in DNA methylation, histone H4 acetylation, andchromosome replication patterns which could arise as a consequenceof the duplication of the whole chromosome set of the originaldiploid genome. Similarly, the number of silver-positive nucleoliroughly correlates to the ploidy level. Early replication andH4 hyperacetylation have been detected at all subterminal chromosomeregions. This, together with cDNA in situ hybridization patterns,indicates the localization of gene-rich regions. DNA methylationand chromosome replication patterns, but not histone H4 acetylation,show differences among the four X chromosomes present: one tothree X chromosomes were observed as hypermethylated and/orlate replicating. Taken together, the data demonstrate thatthere is no overall silencing of the additional two sets ofautosomes in the tetraploid cells, but the X chromosomes couldbe subject to an irregular dosage compensation. Copyright 1999Annals of Botany Company DNA methylation, histone acetylation, polyploidy, replication patterns, sex chromosomes, Silene latifolia (syn.Melandrium album ).     

厚壳贻贝养殖群体与野生群体线粒体DNA的遗传分析(英文）

采用线粒体DNA COI基因序列对厚壳贻贝2个养殖群体与2个野生群体的遗传多样性进行了研究。4个群体共获得30个单倍型。结果显示：在养殖群体中单倍型的数量和遗传多样性要比野生群体的低,可能是由于只有少量的有效父母本对养殖群体的遗传变异有贡献所致。养殖群体与当地野生群体之间也未发生显著的遗传分化,可能是因为它们之间存在基因流。在今后厚壳贻贝养殖过程中,本研究可以用在对养殖群体进行遗传监测,从而保证养殖群体的遗传多样性水平。     

The Role of DNA Synthesis During Hypocotyl Elongation in Light and Dark

Fluorodeoxyuridine, an inhibitor of DNA synthesis, did not affectgermination and post-germinative growth in the aerial part oflettuce and Haplopappus gracilis seedlings when grown in thelight. In the dark, however, elongation of the hypocotyl wasinhibited by fluorodeoxyuridine, strikingly in lettuce and onlyslightly in Haplopappus gracilis. This could imply that thecontrolling mechanism of hypocotyl elongation is in some casesrelated to DNA synthesis, either because mitotic processes (oftenlittle taken into account in considering hypocotyl growth) areinvolved in the elongation of hypocotyls only when they aregrown in the dark, or because DNA synthesis affects cell elongationdirectly, or through the production of a greater number of endopolyploidcells in the dark. Using mainly autoradiographic and cytofluorimetricmethods, these possibilities were tested. Besides lettuce (Lactucasativa L. var. Great Lakes) and H. gracilis (Nutt.) Gray, radish(Raphanus sativus L. var. Tondo rosso quarantino) and soybean(Soya hyspida Sieb. and Zucc. var. Tokyo) seedlings were alsostudied. Fluorodeoxyuridine drastically inhibits cell elongation onlywhen it is preceded or accompanied by mitotic or endomitoticevents. Need for DNA synthesis during hypocotyl elongation,as well as during early post-germinative growth, seems to beof particular importance when endomitotic processes are involved. DNA synthesis, elongation, endoreduplication, fluorodeoxyuridine, Haplopappus gracilis (Nutt.) Gray, Lactuca sativa L., Raphanus sativus L., Soya hyspida Sieb and Zucc     

家蚕的基因文库

本文报导了家蚕Bombyx ,pro基因文库的构建。Sau3A 部分酶解的12-20kb家蚕染色体DNA片段被克隆在λEMBL4的BamHl位点,得到重组噬菌体数5.5×10⁵Pfu,超过了建库要求的理论值。进一步鉴定表明：重组噬菌体呈Spi^-表型;插入的DNA.片段各不相同;并巳从库内筛选到含有与蚊虫酮酶趴基因同源顺序的阳性重组体,这些结果显示了基因文库的可靠性。     

Age and growth of planktonic squids Cranchia scabra and Liocranchia reinhardti (Cephalopoda, Cranchiidae) in epipelagic waters of the central-east Atlantic

Statolith microstructure was studied in two abundant planktoniccranchiids, Cranchia scabra (56 specimens, 38–127 mm mantlelength, ML) and Liocranchia reinhardti (34 specimens, 99–205mm ML) sampled in epipelagic waters of the western part of theGulf of Guinea (tropical Atlantic). Growth increments were revealedin ground statoliths of both species. It was possible to distinguishtwo growth zones in statolith microstructure by their colourin reflected light of the microscope: the translucent postnuclearzone and pale white opaque zone. Assuming that growth incrementsin statoliths were produced daily, ages of the largest immatureC.scabra and L.reinhardti were 166 and 146 days, respectively.Both cranchiids are fast-growing squids with growth rates inlength resembling those of juveniles of tropical ommastrephidsand Thysanoteuthis rhombus. Liocranchia reinhardti grows faster:its growth rate in ML is approximately twice that of same-agedC.scabra. The life cycle of both cranchiids consists of twophases. During their epipelagic phase, C.scabra and L.reinhardtifeed and grow rapidly from paralarvae to immature young in theepipelagic waters, attaining 120–130 and 170–200mm ML by ages of 4–5 months, respectively. Then they changetheir life style to a deepwater phase.     

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.