Characterization of an <Emphasis Type="Italic">Eco</Emphasis>RI family of satellite DNA from two species

We have cloned and sequenced a 321bp band of repetitive DNA from Eptesicus fuscus and E. serotinus observed after gel electrophoresis of EcoRI digested genomic DNA in both species. Southern blot analysis of genomic DNA (from both species) digested with the same enzyme showed the existence of a ladder pattern indicating that the repetitive DNA is arrayed in tandem. The repetitive sequences have a monomer unit of 321bp which is composed of two subunits of 160bp, suggested by the existence of a 160bp band in the ladder of E. fuscus and by the presence of some direct repeats found in the analysis of the consensus sequence. Analysis of the methylation status demonstrated that cytosines in CCGG sequences in this satellite DNA are methylated in E. fuscus but not in the E. serotinus. Alignment of the sequenced clones showed that several nucleotide positions are diagnostic species-specific and consequently the phylogenetic analysis grouped the monomer units from both species in two clearly separated groups.     

DNA fingerprinting of Peronospora parasitica,a biotrophic fungal pathogen of crucifers

The fungus Peronospora parasitica (Pers. ex Fr.) Fr. is an obligate biotroph infecting a wide range of host species in the family Cruciferae. Isolates from different hosts are morphologically similar, and pathotypes are usually distinguished on the basis of host range. Random Amplified Polymorphic DNA (RAPD) fingerprints were generated from a range of P. parasitica isolates from different Brassica species. Reaction conditions, in particular DNA template, primer and Mg²⁺ concentrations, were optimized to ensure that amplifications were reproducible. Possible artefacts arising through host plant DNA were assessed by including such DNA in control reactions. Confirmation that diagnostic RAPD bands were generated from fungal DNA was also obtained by Southern hybridization of a RAPD band to genomic fungal DNA. By screening 20 decamer primers, 2 were found to detect sufficient genetic variation to allow complete differentiation between pathotypes. These results illustrate the potential value of RAPDs for detecting polymorphisms between isolates of a non-culturable plant pathogenic fungus.     

Use of a 'Miniprep' for rapid extraction of plasmids from vancomycin- and gentamicin-resistant Enterococcus faecium

Conventional methods of plasmid extraction are largely unsuited to diagnostic laboratories. The 'Miniprep' is a rapid method that utilises a centrifugable column to separate plasmid DNA from chromosomal DNA. We have modified this technique to extract plasmid DNA from seven strains of vancomycin- and gentamicin-resistant Enterococcus faecium (VGREF): 1% mannitol was added to the growth medium and cell lysis was achieved by incubation in 10 mg of lysozyme/ml in 10 mM Tris, 1 mM EDTA and 25% sucrose at pH 8.0. RNase A was added to plasmid eluate rather than at the lytic step. In comparison to a standard phenol/chloroform method, Miniprep completely eliminated chromosomal interference in gel electrophoresis but otherwise produced identical plasmid profiles. Plasmids obtained from the VGREF ranged from 42 to 1.3 Md. Band densities on a single elution from the Miniprep varied from 8.3 to 106.3 relative units. Double elution increased band densities from the same preparation from 30.4 to 196 relative units; mean percentage increases per track between 7.0 and 34.6%. This method is suitable to achieve plasmid DNA extraction from VGREF within 1 h, making the process more suitable for diagnostic laboratories.     

Assessment of DNA fingerprinting for determining genetic diversity in sheep populations

Genomic DNA, prepared from 12 animals from four sheep flocks, was digested with either HaeIII or Hin fI and probed with three DNA fingerprinting probes. Mean DNA fingerprint band sharing and band frequency calculated for each flock were used to estimate genetic diversity. Each of the DNA fingerprinting systems showed the same trend in diversity within the sampled flocks, and greater diversity between the flocks than within the flocks. DNA fingerprinting therefore provides a useful measure of genetic diversity in sheep.     

Effects of Ethidium Bromide and Several Acridine Dyes on the Kinetoplast DNA of Leishmania tropica*†

Whole cell DNA from Leishmania tropica has 2 peaks when banded by CsCl equilibrium density centrifugation. The main band has a buoyant density of 1.721 and the satellite band a buoyant density of 1.705, with Clostridium perfringens DNA (ρ= 1.6915) used as a reference. The satellite band has been identified as the kinetoplast DNA by purifying DNA from isolated kinetoplasts. L. tropica has the highest G + C content of both nuclear and kinetoplastic DNA thus far reported for trypanosomatids. The effects of ethidium bromide, acriflavin, proflavin, and 5-aminoacridine on the kinetoplast of L. tropica have been compared. Ethidium bromide and acriflavin, but not proflavin or 5-aminoacridine, induce dyskinetoplasty. L. tropica is one of the most sensitive trypanosomatids to ethidium bromide and acriflavin. Examination of the DNA from drug-treated cells in CsCl gradients revealed a loss of the satellite band after ethidium bromide or acriflavin treatment, but not after proflavin or 5-aminoacridine treatment. Cell division was required to produce these effects on the kinetoplast.     

Design and Evaluation of a Specific Primer Pair for the Diagnosis and Identification of <Emphasis Type="Italic">Acanthamoeba polyphaga</Emphasis>

Random amplified polymorphism DNA (RAPD) is a useful tool for species identification. The obtained band patterns can be used for specific primer pair design that may be useful for species diagnosis. In this study, a distinctive a 962-bp band in A. polyphaga band patterns was found, by using the OPC20 primer (ACTTCGCCAC). The DNA fragment was used to design a specific primer pair that was useful for the identification of different isolates as A. polyphaga species. A case of A. polyphaga in disseminated acanthamoebiasis affecting mesenteric nodes is also reported.     

Analysis ofDrosophila chromosome4 using pulsed field gel electrophoresis

Previous estimates of the size ofDrosophila melanogaster chromosome4 have indicated that it is 1% to 4% of the genome or 6 Mb. We have used pulsed field gel electrophoresis (PFGE) to separate megabase-sized molecules ofD. melanogaster chromosomal DNA. Southern blots of these gels were probed with DNA fragments from thecubitus interruptus andzfh-2 genes, which are located on chromosome4. They each identify the same-sized distinct band that migrates at approximately 5.2 Mb in DNA preparations from the Kc cell line. We interpret this band to be intact chromosome4. In DNA obtained from embryos of variousD. melanogaster wild-type strains, this chromosome band showed strain-specific size variation that ranged from 4.5 to 5.2 Mb. TheD. melanogaster chromosome4 probes also identified a single, 2.4 Mb band in embryonic DNA fromDrosophila simulans. We conclude thatD. simulans chromosome4 is substantially smaller than that ofD. melanogaster, presumably owing to diffirences in the amount of heterochromatic DNA sequences. Our simple DNA preparation from embryos and PFGE conditions should permit preparative isolation of chromosome4 DNA and will facilitate the molecular mapping of this chromosome.     

Superhelix density heterogeneity in closed circular intracellular PM2 DNA

Covalently closed intracellular DNA obtained from Pseudomonas BAL 31 20 min after infection with PM2 phage has been shown to be heterogeneous in superhelix density. Analytical band sedimentation, in the presence of low concentrations of ethidium bromide, has been carried out on fractions centripetal and centrifugal to the mode of a single band of closed circular DNA in a preparative propidium iodide–CsCl buoyant density gradient. Different average sedimentation rates, as well as different band shapes, have been observed for upper and lower fractions centrifuged at a dye concentration near the minimum in s° versus ethidium bromide concentration titrations performed on DNA from proximate intermediate fractions. Similar differences, although not as pronounced, have been obtained at a dye concentration corresponding to a point in the steep region of the titrations. Differential band sedimentation experiments performed on the same fractions have confirmed these results. Differential band sedimentation experiments on similarly fractionated mature PM2 I DNA (closed circular form) have shown slight differences in the relative sedimentation rates of upper and lower fractions at dye concentrations corresponding to the steep regions in the titrations. The same experiments, when performed on nicked circular DNA obtained from heating both the mature and intracellular fractions, showed no evidence of differences in sedimentation coefficients. Such results may indicate slight heterogeneity in the superhelix density of viral PM2 I DNA; however, the degree of this heterogeneity would be somewhat less than that of the intracellular DNA. The possibility that superhelix density heterogeneity may arise from displacement loops, which have been found at low levels in intracellular PM2 DNA, has been subjected to experimental tests. Unless such structures are originally present and removed by the isolation procedure, this possibility may be rejected.     

Male-specific DNA markers from African catfish (Clarias gariepinus)

We searched for sex-specific DNA sequences in the male and female genomes of African catfish, Clarias gariepinus (Burchell, 1822) by comparative random amplified polymorphic DNA (RAPD) assays performed on pooled DNA samples. Two sex-linked RAPD markers were identified from the male DNA pool and confirmed on individual samples, showing good agreement with phenotypic sex. Both markers were isolated, cloned and characterized. The first marker (CgaY1) was nearly 2.6 kb long, while the length of second one (CgaY2) was 458 bp. Southern blot analysis with a CgaY1 probe showed strong hybridizing fragments only in males and not in females under stringent conditions, indicating the presence of multiple copies of CgaY1 in the male genome. When tested by zoo blot on the genomes of two closely related species from the Clariidae family, CgaY1 hybridized to the DNA of Heterobranchus longifilis and generated a faint male-specific band at low stringency. CgaY2 produced similar hybridization pattern in both sexes of C. gariepinus, C. macrocephalus and H. longifilis. Specific primers were designed to the sequences and the markers were amplified in multiplex PCR reactions together with a control band common to all individuals. This allowed for rapid, molecular sexing of the species on the basis of a simple three band (male) versus one band (female) pattern. According to our knowledge these are the first sex-specific DNA markers isolated from a siluroid fish species. This revised version was published online in July 2006 with corrections to the Cover Date.     

Temperature dependence of CD spectra of DNA from various sources

The CD spectra of DNA from various sources (T2; T4; C_d; Escherichia coli; calf thymus; Streptomyces chrysomalis) were investigated. A new band Δ_ε210 in the CD spectra of glucosylated DNA of the T even phages was found. The temperature dependence of the CD spectra of DNA was obtained over a wide range of temperatures, including those of the helix–coil transition. The band Δ_ε275 for all DNAs does not appreciably change in the range of the helix–coil transition. The monotonic increase of this band before melting, and its decrease after melting is observed with an increase in temperature. The amplitude of the CD band Δ_ε245 for all the DNAs studied and Δ_ε210 (glucosylated DNA) parallels the change of E₂₆₀ absorbance.     

Chromatin structure of Drosophila nasutoides satellites as probed by cross-linking DNA in situ

Chromatin structure can be probed by cross-linking DNA in situ using trioxsalen and irradiation with UV light. Presumably DNA within a nucleosome is protected from cross-linking so that this region appears as a single-strand loop in the electron microscope under a condition in which single-strands and double-strands are distinguished. Unprotected regions appear as duplex due to cross-linking.We have used this approach to investigate the structure of chromatins containing satellite DNAs of Drosophila nasutoides. We have previously shown that D. nasutoides has an unusually large autosome pair which is almost entirely heterochromatic. Its nuclear DNA reveals four major satellite components amounting up to 60% of the total genome. All of them are localized in this large heterochromatic chromosome. We wish to ask whether chromatins containing different satellite sequences have different arrangements of nucleosomes. Our results from cross-linking experiments show that all DNA components including main band DNA have different patterns of protected and unprotected regions: (a) The length distributions of protected regions show multiple peaks with the smallest unit lengths being 200 nucleotides for main band DNA, 180 for satellites I, II and III, and 160 for satellite IV. (b) The amounts of unprotected regions, presumably internucleosome DNA, vary from 16% for main band DNA to 60% for satellite IV, suggesting that satellite chromatins have fewer nucleosomes per given length of chromatin than main band DNA chromatin. The spacings between nucleosomes appear to be random in satellite chromatins.     

Use of fragment–sharing estimates from DNA fingerprinting to determine relatedness in a tropical wren

We assessed the usefulness of DNA fragment–sharing scores from DNA fingerprints for assigning relatedness to unknown pairs of individuals in a population of stripe–backed wrens (Campylorhynchus nuchalis). Preliminary investigation of scoring biases revealed consistency both within and between scorers in relative band–sharing scores, but a tendency for scores to be inflated and for inter–scorer agreement to decline as distance between lanes on an autoradiograph increased. Distributions of band–sharing values matched expected distributions well, which suggests that variability in scores is mostly inherent and not a result of errors in scoring. Confidence intervals based on band–sharing scores or means of scores across enzymes, probes and scorers revealed that unrelated (r= 0) and first–order dyads (r=Vi) could be distinguished on the basis of single band–sharing scores from the best combination of enzyme and probe (HaeIII/33.15) and that first– and second–order dyads could be distinguished when confidence intervals were based on means of band–sharing scores across two enzymes, two probes and two scorers.     

Identification of molecular markers in soybean comparing RFLP,RAPD and AFLP DNA mapping techniques

Three different DNA mapping techniques—RFLP, RAPD and AFLP—were used on identical soybean germplasm to compare their ability to identify markers in the development of a genetic linkage map. Polymorphisms present in fourteen different soybean cultivars were demonstrated using all three techniques. AFLP, a novel PCR-based technique, was able to identify multiple polymorphic bands in a denaturing gel using 60 of 64 primer pairs tested. AFLP relies on primers designed in part on sequences for endonuclease restriction sites and on three selective nucleotides. The 60 diagnostic primer pairs tested for AFLP analysis each distinguished on average six polymorphic bands. Using specific primers designed for soybean fromEco RI andMse I restriction site sequences and three selective nucleotides, as many as 12 polymorphic bands per primer could be obtained with AFLP techniques. Only 35% of the RAPD reactions identified a polymorphic band using the same soybean cultivars, and in those positive reactions, typically only one or two polymorphic bands per gel were found. Identification of polymorphic bands using RFLP techniques was the most cumbersome, because Southern blotting and probe hybridization were required. Over 50% of the soybean RFLP probes examined failed to distinguish even a single polymorphic band, and the RFLP probes that did distinguish polymorphic bands seldom identified more than one polymorphic band. We conclude that, among the three techniques tested, AFLP is the most useful.     

Satellite DNA associated with heterochromatin in Rhynchosciara

The DNA of Rhynchosciara hollaenderi was examined using isopycnic centrifugation in neutral CsCl. Two low density minor bands (collectively termed satellite DNA) were detected in addition to the main band DNA. Main band DNA has a buoyant density of 1.695 g/cm³. The larger of the two minor bands has a buoyant density of 1.680 g/cm³ while the smaller of the two minor bands has a buoyant density of about 1.675 g/cm³. Thermal denaturation studies have confirmed the presence of the two minor classes of DNA.—The satellite and main band DNAs were isolated in relatively pure form and were transcribed in vitro using DNA-dependent RNA polymerase from Escherichia coli. Annealing of the two complementary RNAs (cRNAs) with main band and satellite DNA was examined using filter hybridization techniques.—The chromosomal distribution of the satellite DNA was determined by in situ molecular hybridization of satellite-cRNA with Rhynchosciara salivary gland chromosomes. Satellite-cRNA hybridized with the centromeric heterochromatin of each of the four chromosomes (A, B, C, and X) and with certain densely staining bands in the telomere regions of the A and C chromosomes. Main band-cRNA annealed with many loci scattered throughout the chromosomes including areas containing satellite DNA.     

Synthesis and characterization of amplified DNA in oocytes of the house cricket,Acheta domesticus (Orthoptera: Gryllidae)

At a time in the life cycle when a large proportion of the oocytes of Acheta incorporate ³H-thymidine into an extrachromosomal DNA body, synthesis of a satellite or minor band DNA, the density of which is greater than main band DNA, is readily detected. Synthesis of the satellite DNA is not detectable in tissues, the cells of which do not have a DNA body, or in ovaries in which synthesis of extrachromosomal DNA by the oocytes is completed. The DNA body contains the amplified genes which code for ribosomal RNA. However, less than 1 percent of the satellite DNA, all of which appears to be amplified in the oocyte, is complementary to ribosomal 18S and 28S RNA. In situ hybridization demonstrates that non-ribosomal elements, like the ribosomal elements of the satellite DNA, are localized in the DNA body.Abbreviations used rRNA ribosomal RNA, includes 18S and 28S RNA - rDNA gene sequences complementary to rRNA - cRNA complementary RNA synthesized in vitro     

Circular dichroism of histone-bound regions in chromatin.

Native, NaCl-treated, trypsin-treated, and polylysine-bound nucleohistones were studied in 2.5 × 10^?4 M EDTA, pH 8.0, using circular dichroism (CD) and thermal denaturation. Removal of histone I by 0.6 M NaCl has a much smaller effect on both Δε₂₂₀ and Δε₂₇₈ than the removal of other histones. This indicates that histone I has less helical content and less conformational effect on the DNA in nucleohistone. By extrapolating to 100% binding by histones other than I, the positive CD band near 275 nm is close to zero. Comparison is also made between the effects of binding by the more basic and the less basic halves of histones by trypsin-digestion and polylysine-binding experiments. Trypsin digestion of nucleohistone reduces melting band IV at 82°C much more than melting band III at 72°C. However, the CD changes of Δε₂₇₈ and Δε₂₂₀ induced by trypsin digestion are small, unless melting band III is also reduced by the use of a higher trypsin level. This implies that the less basic halves of histones, which stabilize DNA to 72°C (melting band III), have more helical structure and are more responsible for conformational change in DNA than are the more basic halves, which stabilize DNA to 82°C (melting band IV). Polylysine binding to nucleohistone diminishes melting band III but has no effect on melting band IV. This binding affects only slightly the Δε₂₂₀ of nucleohistone, indicating that polylysine interferes very little with the structure of the less basic halves of bound histones. The implications of these studies with respect to chromatin structure are discussed.     

Quasielastic light scattering by biopolymers. IV. Tertiary collapse of calf thymus DNA in 5.5M LiCl

Circular dichroism has been commonly employed to infer the conformation of DNA in solution. The basis of the conformational assignments is the work of Tunis-Schneider and Maestre, wherein CD spectra of DNA were obtained under conditions comparable to those employed in the x-ray diffraction studies of A-, B-, and C-DNA. It has recently been suggested that the CD spectrum of DNA in chromatin, which is similar to the CD spectrum of the C-form DNA, is a superposition of the normal B-DNA spectrum and a single negative band, centered at 275 nm. This negative band is qualitatively identical to the spectrum for condensed Ψ-form DNA. We have employed the hydrodynamic methods of quasielastic light scattering and sedimentation velocity to determine the extent of DNA tertiary structural alteration in 5.5M LiCl as a possible explanation of the C-form CD spectrum. These studies suggest an eightfold contraction of the Stokes hydrodynamic volume for calf thymus DNA in going from 0.4M NH₄Ac to 5.5M LiCl, with no change in molecular weight. The estimated maximum presistence length of DNA in 5.5M LiCl is estimated to be 20.0 nm compared to the “minimum” value of 44.7 nm in NaCl solutions. The value 20.0 nm corresponds to a maximum radius of 16.7 nm for a “continuously coiled” cylinder of DNA, which compares with the value 5.0 nm of DNA in the nucleosome unit of chromatin.     

Linear dichroism studies of nucleic acids. II. Calculation of reduced dichroism curves of A-and B-form DNA

We have calculated the uv linear dichroism for the A- and B-forms of DNA using π-π* transition moments and band components determined from the free DNA bases. The reduced dichroism (LD^R) as a function of wavelength is estimated is in the 220–300-nm region, for both the oriented-gas model and a simple exciton model. For B-form DNA, LD^R is obtained to ?1.48S (S being the orientation factor) over the whole wavelenth region by both models. For A-form DNA, LD^R is not constant, but changes monotonically from about ?1.15S at 220 nm to about ?1.35S to ?1.45S at 300 nm, depending on base combination and degree of interaction (?1.35S for the oriented gas). It is emphasized that a common assumption of a single “effective” transition moment of the principal band at 260 nm may not generally be made because of the extensive overlap of differently polarized bands. The possibility of using the reduced dichroism curve for characterizing the secondary structure of DNA is discussed.     

Alcune caratteristiche strutturali dei cromosomi politenici del sospensore embrionale di Phaseolus coccineus in due momenti dell'embriogenesi

Abstract

Some features of polytene chromosomes of Phaseolus coccineus suspensor during two stages of early embryogenesis. – The distribution of DNA and RNA puffs in the whole genome of the giant cells of the Phaseolus coccineus embryo suspensor has been detected in two stages of embryo development. The collected data show that the chromosome regions showing the highest frequency of DNA puffs in both analysed stages are the following: i) band B (the fraction proximal to secondary constriction) of chromosome pair I and band E of chromosome pair V. When the two stages of development are however compared, it is seen that the % of DNA puffs in chromosome pair is at least double in suspensors dissected from the first stage of embryo development (86% in the first stage; 41% in the second stage). As to chromosome pair V band E organizes DNA puffs in 36% and in 50% of observed chromosomes in the first and second stage respectively; ii) band A of chromosome pair II, with a frequency of 52% (first stage) and 27% (second stage); iii) band E of chromosome pair VIII (27% in the first stage and 19% in the second stage). As far as the organization of RNA puffs is concerning it seems possible to outline the following values as the highest percentages:

a) First stage. chromosome b) Second stage. chromosome

I: band B 83% I: band B 91% VI: band E 55%

II: band A 70% II: band A 51% band G 50%

IV: band E 45% band C+D 59% VIII: band E 44%

V: band B+C 57% IV: band E 51% IX: band A 54%

band E 71% band G 51% band E 43%

VI: band E 53% band I 51% band F 53%

VIII: band E 43% V: band E 43%

IX: band E 42%

The differences observed between the two stages are discussed in relation to the function of the suspensor.     

Evaluation of a reverse-hybridization StripAssay for the detection of genetic polymorphisms leading to acenocoumarol sensitivity

Acenocoumarol is mainly catabolized by CYP2C9 isoform of cytochrome P450 (CYP) liver complex and exerts its anticoagulant effect through the inhibition of Vitamin K Epoxide Reductase (VKOR). The most important genetic polymorphisms which lead to an impaired enzymatic activity and therefore predispose to acenocoumarol sensitivity, are considered to be CYP2C9*2 (Arg144Cys), CYP2C9*3 (Ile359Leu) and VKORC1-1639G>A, respectively. In this study we compared the results of the PGXThrombo StripAssay kit (ViennaLab Diagnostics,Vienna, Austria) with direct DNA sequencing and in house Restriction Fragment Length Polymorphisms (RFLP) for the detection of the aforementioned Single Nucleotide Polymorphisms (SNPs). The reverse hybridization StripAssay was found to be equally effective with RFLP and direct DNA sequencing for the detection of CYP2C9*2 and CYP2C9*3 polymorphisms, respectively. The comparison of the RFLP reference method with the reverse hybridization StripAssay for the detection of VKORC1-1639 G>A polymorphism showed that the reverse hybridization StripAsssay might misclassify some A/A homozygotes as heterozygotes. Optimization of the hybridization procedures may eliminate the extra low signal band observed in some samples at the reverse hybridization StripAssay and improve its diagnostic value.