Physical properties of endonuclease S1 digestion products of DNA renaturation intermediates.

The growth of the weight-average double-strand length of renaturing sonicated DNA was investigated as a function of the degree of renaturation. Endonuclease S1 was used to digest single-strand ends and gaps. The ratio of the average renatured length to the initial length was found to be 0.6 ± 0.1 early in the reaction and to approach 1.0 at infinite time, while the ratio of the average single-strand length of native regions increased from 0.6 ± 0.1 to 0.8 ± 0.1 at infinite time. The ratio increases are nonlinear, with the greatest growth occuring around 50% renaturation. Relaxation times of double-strand DNA were measured in low salt using alternating field electric birefringence and were found to increase to only 70% of the relaxation time of the initial DNA at infinite time. In the molecular-weight range examined (150–320,000 daltons), relaxation times vary with the 2.3 ± 0.3 power of molecular weight.     

Use of a DNA probe for mapping by electron microscopy the ribosomal sequences in ribosomal RNA precursors from duck cells

This report describes the use of purified ribosomal DNA to map by electron microscopy the relative positions of the 18 S and 28 S RNA regions within the duck rRNA precursor and their relationship to the non-conserved portions of the precursor molecule. By repeated fractionation of the total DNA, based on the relative reassociation rates of the DNA sequences with different degrees of repetition, a fraction of the rapidly renaturing DNA was obtained which comprised only 6% of the total DNA, but contained 71% of the rRNA cistrons. Further purification of the rDNA was achieved by saturation hybridization with rRNA and separation of the rRNA-rDNA hybrids by banding in CsCl. In this manner, an rDNA-rRNA fraction was obtained which had a buoyant density of 1.805 g/cm³, an RNA to DNA ratio of 1.01, and a base composition for the RNA present in the hybrid identical to that of an equimolar mixture of 18 S and 28 S rRNA. The final yield of rDNA isolated by this procedure is 32%. When the purified rDNA was annealed with a mixture of 18 S and 28 S rRNA and the hybrids spread for electron microscopy, they appeared as two distinct populations with a number-average length of 0.62 ± 0.13 μm and 1.37 ± 0.18 μm, respectively. Likewise, hybrids between the rRNA precursor, isolated from duck embryo fibroblasts, and the rDNA appeared as structures containing two duplex regions of lengths 0.60 ± 0.11 μm and 1.38 ± 0.15 μm, separated from each other by a single-stranded region appearing as a large bush: this represents a portion of the precursor molecule not conserved during processing of the parent molecule. From these observations a model of the structure of the duck rRNA precursor is proposed.     

The sequence arrangement of Drosophila melanogaster 5s DNA cloned in recombinant plasmids.

Segments of Drosophila melanogaster DNA containing 5S rRNA genes have been propagated in recombinant plasmids using E. coli as a host and Col E1 as a vector. Electron microscope partial denaturation mapping, mapping by ferritin labeling and restriction enzyme-gel electrophoresis analysis all indicate that the Drosophila DNA inserts of these plasmids consist of tandem repeats of 5S genes and spacer regions. The repeat length is approximately 380 nucleotide pairs (ntp), corresponding to a gene of length 120 ntp and a spacer of length 260 ntp. The insert in one plasmid (pCIT9) consists of 32 contiguous repeats. Restriction enzyme-gel electrophoresis analysis shows that all these repeats have the same length within ± 5 nucleotides. This repeat length is estimated as 370 ± 20 ntp by gel electrophoresis and 390 ± 40 ntp by partial denaturation mapping. A second plasmid (pCIT19) contains three complete genes, two complete spacers and incomplete flanking spacer sequences. The two complete repeat units released by suitable restriction endonuclease digestions differ in length by 20 ± 5 ntp, with estimated lengths of 370 and 390 ntp. The positions and spacings of the genes on this plasmid have been observed directly by ferritin labeling and by partial denaturation mapping. The A+T content of the 5S DNA spacer region is calculated to be 68%. By in situ hybridization, cRNA transcribed from one plasmid hybridizes to polytene chromosomes only at band 56F, the known locus of the 5S rRNA genes. Spontaneous excision of some of the tandem repeat units from the recombinant plasmids occurs during growth in E. coli; the frequency of excision does not depend upon the recA character of the host, but is greatly increased by chloramphenicol treatment.     

Different arrangements of protein subunits and single-stranded circular DNA in the filamentous bacterial viruses fd and Pf1

A single-stranded circular DNA molecule of 6690 ± 450 nucleotides accounts for 5.5 ± 0.3% of the mass of Pf1 virus. The remaining mass is contributed almost entirely by subunits of the major coat protein. A non-integral nucleotide to subunit ratio of 0.87 ± 0.05 is calculated from the DNA content, the average nucleotide mass (309), and the known mass of one protein subunit (4609). There are therefore 7690 ± 680 major coat protein subunits in the virus. The virus length determined by electron microscopy is 1960 ± 70 nm. The data give an average axial distance of 2.55 ± 0.24 Å between protein subunits in dry virus. Since there is an up strand and a down strand of the circular DNA within the virus filament, an axial distance between bases in a given strand of 5.9 ± 0.5 Å is calculated. Available X-ray data show that an axial repeat of 72 Å, or slightly less, would be expected for dry Pf1 virus (0% relative humidity). A structural model in which 27 protein subunits and 24 nucleotides are contained in this repeat would be consistent with our data. The DNA conformation and the subunit packing in Pf1 differ considerably from those in fd, even though both are filamentous viruses containing single-stranded circular DNA. The uncertainties cited are 95% confidence limits.     

Unique arrangement of coding sequences for 5 S, 5.8 S, 18 S and 25 S ribosomal RNA in Saccharomyces cerevisiae as determined by R-loop and hybridization analysis.

The arrangement of the coding sequences for the 5 S, 5.8 S, 18 S and 25 S ribosomal RNA from Saccharomyces cerevisiae was analyzed in λ-yeast hybrids containing repeating units of the ribosomal DNA. After mapping of restriction sites, the positions of the coding sequences were determined by hybridization of purified rRNAs to restriction fragments, by R-loop analysis in the electron microscope, and by electrophoresis of S₁ nuclease-treated rRNA/rDNA hybrids in alkaline agarose gels. The R-loop method was improved with respect to the length calibration of RNA/DNA duplexes and to the spreading conditions resulting in fully extended 18 S and 25 S rRNA R-loops. The qualitative results are: (1) the 5 S rRNA genes, unlike those in higher eukaryotes, alternate with the genes of the precursor for the 5.8 S, 18 S and 25 S rRNA; (2) the coding sequence for 5.8 S rRNA maps, as in higher eukaryotes, between the 18 S and 25 S rRNA coding sequences. The quantitative results are: (1) the tandemly repeating rDNA units have a constant length of 9060 ± 100 nucleotide pairs with one SstI, two HindIII and, dependent on the strain, six or seven EcoRI sites; (2) the 18 S and 25 S rRNA coding regions consist of 1710 ± 80 and 3360 ± 80 nucleotide pairs, respectively; (3) an 18 S rRNA coding region is separated by a 780 ± 70 nucleotide pairs transcribed spacer from a 25 S rRNA coding region. This is then followed by a 3210 ± 100 nucleotide pairs mainly non-transcribed spacer which contains a 5 S rRNA gene.     

DNA heteroduplex analysis of the relation between bacteriophages phiX174 and S.13

When observed in the electron microscope using the isodenaturing methods of Davis &; Hyman (1971), only one small segment (4.7 ± 1.9%) of the DNA of phage φX174 is highly homologous with phage S.13 DNA; the rest is partially homologous with an over-all average 36% base mismatch. The two phage DNA molecules appear to be identical in length and have no regions of complete base non-homology. The phage-coded proteins were compared by electrophoresis on slab polyacrylamide gels and only one of the S.13 coded proteins migrated identically with its φX174 counterpart. The other eight S.13 coded proteins varied in size from their φX174 counterparts by +4.6% to ?6.0% (± ten amino acid residues). The relevance of these data to the complementation and recombination between these two phages is discussed.     

Evaluation of copper and vanadium concentration in the soft tissue of Tylosurus crocodilus in Bahregan region,Persian Gulf

Abstract

The present study has been carried out to evaluate the concentration of copper (Cu) and vanadium (V) elements in the muscle, skin, and gill tissues of Tylosurus crocodilus (Belonidae), an edible fish, in the Bahregan region which is located in the northern part of the Persian Gulf. For this purpose, 18 fish samples in three different length groups (small, medium, and large) were randomly collected in Bahregan region. Tissues were separated and concentration of the heavy metals was determined by atomic absorption. The results showed that mean concentration of Cu and V were 0.2851 ± 0.10036 and 1.1368 ± 0.12405, 0.1117 ± 0.02322 and 2.5091 ± 1.14201 and 0.1803 ± 0.03204 and 3.3133 ± 1.05995 μg g^-1 dry weight, in skin, muscle and gill tissues, respectively. Also, the results revealed a significant positive correlation between the standard length and Cu concentration in the muscle and gill tissues (P ≤ 0.05), as well as between the standard length and V concentration in all the tissues (P ≤0.05). The results indicated that the concentration of V was higher than the World Health Organization (WHO) standard but the concentration of Cu was lower. It is therefore highly recommended that usage of this carnivore species in the studied area is limited because of the high accumulation of V in its muscle tissue.     

Heterocyst division in two blue-green algae

Bacteriophage 16-6-12 of Rhizobium lupini has a long, non-contractile tail and a head which is hexagonal in outline. The tail is 140 nm in length, 11 nm in diameter, and carries a short terminal fiber. Analysis of the tail structure by optical diffraction indicates that it is of the helical “stacked disc” type. After phenol-extraction from purified particles, the DNA of phage 16-6-12 can circularize in vitro. No significant difference in contour length was observed between the linear (14.34±0.28 μm) and circular (14.44±0.24 μm) forms of molecules. After partial denaturation with alkali an AT-GC-map was constructed, which shows an asymmetric distribution of AT- and GC-rich regions. It is concluded that this phage DNA can circularize due to the presence of cohesive ends and that it is not circularly permuted.     

In vitro propagation and assessment of genetic integrity of Talinum triangulare (Jacq.) Willd: a valuable medicinal herb

Talinum triangulare is a medicinally important herb and various parts of the plant are used pharmaceutically for the treatment of different diseases. In our study, a rapid and efficient protocol for micropropagation has been developed from shoot tip and nodal explants of T. triangulare. High shooting frequency (93.33?%) was achieved with shoot tip explants when cultured on Murashige and Skoog??s (MS) medium supplemented with 1.0?mg/L 6-benzyl amino purine (BAP) producing an average of 12.50?±?0.23 shoots and 5.07?±?0.02?cm shoot length per explant. A combination of 0.5?mg/L BAP and 0.5?mg/L kinetin was found to be more effective by producing 15.67?±?0.25 shoots and 6.22?±?0.02?cm shoot length per explant. The microshoots were excised and cultured on half-strength MS and full-strength MS medium containing different concentrations of indole-3-acetic acid and indole-3-butyric acid (IBA) for root induction. More number of roots (45.10?±?0.96) with an average length of 5.46?±?0.08?cm was obtained on half-strength MS medium supplemented with 0.5?mg/L IBA. The rooted shoots were successfully transplanted from different planting substrates to the field with a 100?% survival rate. Random amplified polymorphic DNA analysis was carried out using four random decamer primers. The amplification products were monomorphic in the micropropagated plants and were similar to the mother plant. Absence of polymorphism revealed that no variation was induced, thus maintaining the genetic integrity of the micropropagated plants of T. triangulare.     

PCR-based evaluation of sequence specificity of DNA fragmentation by ultrasound

Ultrasonic fragmentation, which is a simple and convenient method for the mechanical degradation of DNA, is widely used in modern genome studies as one of the sample preparation steps. It has been recently found that the DNA breaks occur more often in the regions containing 5'-CG-3' dinucleotides. We studied the influence of the 5'-CG-3' dinucleotides on the efficiency of the 28S rRNA gene amplification during PCR with sonicated DNA of Mantis religiosa. It was shown that the amplification rate depends on the template length and the number of 5'-CG-3' dinucleotides. Amplification of the DNA regions with a higher 5'-CG-3' density is less efficient because of their higher sensitivity to ultrasound. The amount of the amplified DNA templates is inversely proportional to the 5'-CG-3'number.     

Cloning,characterization and chromosomal location of a satellite DNA from the Pacific oyster,Crassostrea gigas

We report the cloning and characterization of a high-copy-number, tandem-repeat satellite DNA sequence from the genome of the Pacific oyster, Crassostrea gigas (Cg). The monomeric unit was found to be 166 (±2) bp in length with 79–;94% homology between monomers of the array. The sequence is A+T-rich (60%) and lacks internal repetition and substructural features. The repeat was estimated to account for 1–;4% of the Cg genome. Fluorescence in situ hybridization (FISH) studies mapped the repeat to two distinct heterochromatic regions of two pairs of homologous chromosomes on Cg embryonic metaphases. Also, the number of metaphase chromosomes containing this repeat varied with the ploidy of the cell.     

The role of the ovarian horn locus in regulation of spontaneous electric activity of myometrial rhythmogenic areas

The role of the ovarian area of the uterine horn in coordination of spontaneous activity of myometrial rhythmogenic areas was studied in nonpregnant rats both under normal conditions and following transection of the uterine horn in its middle part to isolate the ovarian locus from the distally located uterine active areas. The effect of oxytocin as a factor that reveals a leading role of the ovarian locus in synchronization of myometrial spontaneous activity was studied under the above conditions. Intravenous oxytocin administration (10^–1 μg/kg) under normal conditions promotes a considerable increase in the peak amplitude and mean rise rate in all the three rhythmogenic areas (%; ovarian horn area—by 148.63 ± 6.1, p ≤ 0.001 and 141.04 ± 7.6, p ≤ 0.01; cervical horn area—by 143.85 ± 3.5, p ≤ 0.001 and 146.89 ± 8.5, p ≤ 0.001; uterine corpus—by 146.20 ± 7.2, p ≤ 0.001 and 139.73 ± 8.2, p ≤ 0.05, respectively). Under the same conditions, also there is a similar increase in the active state duration in all the three areas (%; 132.70 ± 4.5, p ≤ 0.05; 124.90 ± 9.6, p ≤ 0.05; 128.03 ± 7.2, p ≤ 0.05, respectively). Following transection of the uterine horn, oxytocin administration causes an increase in all the three activity indicators only in the ovarian horn area (%; 134.86 ± 2.5, p ≤ 0.05; 139.49 ± 4.5, p ≤ 0.001; 123.8 ± 7.3, p ≤ 0.05, respectively). In the cervical horn area and uterine corpus, no appreciable changes in these indicators were detected under both conditions. We believe that the ovarian locus is involved in coordination of activities of all the three myometrial rhythmogenic areas as revealed by oxytocin.     

Cooperative lengths of DNA during melting

The mean cooperative length of domains of DNA, determined from the variance in (G + C) content in derivative melting curves of large bacterial DNAs, varies from 230 base pairs (bp) for (A ? T)-rich domains to 580 bp for (G ? C) domains. These values correspond to values for the cooperativity parameter of 2(±2) × 10^?5 and 3(±2) × 10^?6, respectively, and to +7.2 and +9.6 kcal for the free energy of helix interruption in those regions.     

Autonomous rDNA molecules containing single copies of the ribosomal RNA genes in the macronucleus of Tetrahymena pyriformis

The DNA containing the genes for rRNA (commonly called rDNA) of $\text{Tetrahymena}$ sediments in sucrose density gradients considerably slower than the main part of the DNA when DNA from gently lysed whole cells or isolated nuclei are fractionated by this method. In rDNA purified by CsCl gradient centrifugation about 20% of the DNA (40% of the bases in one strand) consists of sequences homologous to 25S and 17S rRNA as determined by DNA-RNA hybridization. The purified rDNA co-sediments in sucrose gradients with Ø29 phage DNA (M.W. = 11 × 10⁶). Examination by electron microscopy of the rDNA demonstrates that the molecules are linear with a length of 5.65 ±0.6 μm corresponding to a molecular weight of 11 × 10⁶.     

Physical mapping of DNA base sequence homologies between an octopine and a nopaline Ti plasmid of Agrobacterium tumefaciens

A detailed physical map of the homologous and non-homologous regions between an octopine (pTiAch5) and a nopaline (pTiC58) Ti plasmid was determined by Southern type hybridization and by electron microscope heteroduplex analysis. This map was correlated with the functional maps of both plasmids. For the Southern type hybridizations, total labelled pTiAch5 DNA was hybridized to Southern blots of restriction fragments from a series of hybrid plasmids containing overlapping segments of the whole TiC58 plasmid. Reciprocal experiments were also carried out. The common sequences between the two plasmids (±30%) are restricted to four major stretches of homology. Analysis of heteroduplexes between pTiAch5 and several hybrid plasmids containing specific regions of pTiC58, and of heteroduplexes between hybrid plasmids derived from pTiC58 and pTiAch5 provided a detailed map of the fine structure of the four major homology regions. Two regions are distributed in the same relative order as compared to a common reference point, and two are inversed. Three regions contain a number of small, mostly asymmetrical substitution loops. Several regions distributed over the common DNA sequences were found to be partially homologous.     

Elongation of duplex DNA by recA protein

recA protein, which is essential for the recombination process in Escherichia coli, was incubated in the presence of 5′-γ-thiotriphosphate with circular plasmid pBR_βG containing small single-stranded gaps. Stable complexes were formed which appear in the electron microscope as fibres with a diameter about five times that of naked DNA. Complex formation appears to be a co-operative process whereby the average rise per base-pair with respect to the fibre axis increases from 3·39 ± 0·08 Å to 5·20 ± 0·18 Å. The elongation of DNA by about 50% is compatible with an unwinding of the double helix and an intercalating mode of binding of recA and/or 5′-γ-thiotriphosphate to DNA.     

Zonal Fractionation of Mammalian Metaphase Chromosomes and Determination of Their DNA Content

Chinese hamster cells in culture were synchronized, collected at metaphase, homogenized to release the chromosomes, and the chromosomes fractionated in a sucrose gradient using a zonal centrifuge with an A12 zonal rotor. Chromosomes in the separated fractions as well as in control metaphase spreads were quantitatively classified into five easily distinguished groups, according to individual measurements of length and centromeric index. For each zonal fraction, chemical determinations were made of the amount of DNA per average chromosome. Using the group compositional data for each fraction, the amount of DNA per average chromosome in each of the groups was then calculated to be: Group I (chromosomes 1, 2)= 1.00 ± 0.14 pgm/chromatid; Group II (chromosomes 4, X, 5) -0.39 ± 0.05 pgm/chromatid; Group III (chromosomes Y, 6, 7, 8)=0.24 ± 0.04 pgm/chromatid; Group IV (chromosomes 9, 10, 11)=0.13 ± 0.004 pgm/ chromatid; and Group V (a small marker in this cell line)=0.06 pgm/ chromatid. These values are in good agreement with the literature values for relative chromosomal DNA content derived from cytospectrophotometric measurements of fuelgen stained hamster metaphase spreads. They indicate that unlike the case for human chromatids the amount of DNA found in hamster chromatids is not directly proportional to the chromatid length.

The larger chromosomes contain more DNA per unit length than smaller chromosomes. The magnitude of this effect is considerably greater than that which may be ascribable to centromeric constriction.