Does DNA alone determine the development of the organism? (the information aspect of the problem)

Two closely related controversial problems are discussed: whether the developmental processes can be reduced to the synthesis of polypeptides encoded in DNA, and whether the information in DNA is equivalent to that in the adult organism. Critically considered are the ideas that DNA is only responsible for the protein synthesis, whereas morphogenesis proceeds independently and according to epigenetic regularities of its own. It is stated that development is the realization of genetic information in which more elementary (molecular) processes unambiguously determine a more complex cellular level which in its turn determines morphogenesis of tissues and organs. Various mechanisms of the appearance of new information in the course of development are considered. The statement is made that new information concerns only some individual characters of the organism, whereas most of information that determines the process of development and the structure of the adult organism is created in the course of evolution, is stored in DNA and inherited.     

On the heterogeneity of the slow reassociating (“unique”) DNA

Summary The slow reassociating fraction of mouse DNA (unique DNA), when allowed to reassociate in 0.14 m sodium phosphate buffer at 50 °C showed a biphasic melting curve with a transition at 78–80 °C. On the basis of this feature, the slow reassociating DNA was separated preparatively into two fractions: unique DNA I and II. Their duplexes showed differences with respect to thermal stability, S1 nuclease resistance and rate of reassociation. About one third of the sequences in each fraction were fraction-specific. The conclusion was drawn that for unique DNA I these should be the low repetitive or single copy related sequences (multigene families) and for unique DNA II—the unrelated single copy sequences or recent families of low repetitive not yet diverged sequences.     

Absorption of DNA in the region of vacuum-uv (3–25 eV)

By means of a device that might be considered a modern version of "Ulbricht's sphere" the absorption spectrum and the photoelectric emission of calf thymus DNA was measured in the region of 3 to 25 eV (400 to 50 nm). A tentative explanation of the general shape of the absorption spectrum and of its 6 maxima is given. The results permit a much better insight into some biologic effects of vacuum-uv to be gained than hitherto possible.     

Effect of spermine on interaction of DNA polymerase α from the loach (Misgurnus fossilis) eggs with DNA

Polyamines (putrescine, spermidine and spermine) cause a marked increase in the activity of the loach Misgurnus fossilis DNA polymerase α on activated (gapped) DNA. The stimulatory effect increases in the order: putrescine, spermidine, spermine. Kinetic analysis shows that spermine does not change the affinity of the polymerase for dTTP, but it decreases the enzyme affinity for DNA. The apparent K_m of the polymerase for activated DNA progressively increases from 14 to 1200 μM (nucleotide), if the concentration of spermine rises up to 2 mM, while V_max reaches a maximum at 0.5 mM spermine and then drops at higher polyamine concentrations. Native calf thymus DNA and especially single-stranded DNA from phage M13 appear to be inhibitors of α-polymerase activity on gapped DNA. Dixon plots suggest simple competitive inhibition of the polymerase activity by single- or double-stranded DNA and absence of cooperativity in the interaction of the polymerase with DNA. Hill-plot analysis is compatible with the interpretation that there is only one DNA binding site on each DNA polymerase α molecule. Spermine, even at low concentrations, decreases sharply the affinity of the enzyme for double-stranded DNA, while the enzyme affinity for single-stranded DNA changes insignificantly. Another result of spermine action is the destabilization of the polymerase-DNA complex. The ratio of the ‘static affinity’ of the enzyme to its ‘kinetic affinity’ decreases 2.2-fold in the presence of 0.5 mM spermine. As a result, the sensitivity of DNA synthesis to 3′-deoxy-3′-aminothymidine 5′-triphosphate and to 1-β-d-arabinofuranosylcytidine 5′-triphosphate decreases in the presence of the polyamine. Both spermine effects, the decrease in the ‘nonproductive binding’ of the polymerase to double-stranded regions in DNA and the destabilization of the polymerase-DNA complex, presumably account for the increase in the activity of the loach α-polymerase on activated DNA.     

Differentiation of the Dolly Varden Char (Salvelinus malma) and the Taranetz Char (Salvelinus taranetzi) Inferred from the PCR–RFLP Analysis of Mitochondrial DNA

Genetic differentiation of two sympatric char species, the Dolly Varden char (Salvelinus malma Walbaum) and the Taranetz char (S. taranetzi Kaganovski), has been studied. Restriction fragment length polymorphism (RFLP) analysis has been used to compare three mitochondrial DNA (mtDNA) fragments (ND1/ND2, ND5/ND6, and Cytb/D-loop) amplified via polymerase chain reaction (PCR). The divergence between S. malma and S. taranetzi inferred from mtDNA nucleotide sequences is 2.8%; between S. leucomaenis and S. taranetzi, 7.1%; and between S. malma and S. leucomaenis, 7.5%. The absence of common haplotypes and the degree of divergence indicate that the Dolly Varden char and the Taranetz char are genetically isolated and confirm that S. taranetzimay be regarded as a separate species.     

DNA synthesis in the imaginal wing discs of the American bollwormHelicoverpa armigera (Hübner)

The effect of two insect growth regulators of plant origin viz. plumbagin and azadirachtin and the ecdysteroids 20-hydroxyecdysone, makisterone A and a phytoecdysteroid on DNA synthesis in imaginal wing discs of day 4 final instarHelicoverpa armigera larvae was studied. DNA synthesis increased with increase in time of incubation up to 8 h and decreased later without the addition of moulting hormone. Addition of 20-hydroxyecdysone supported long term acquisition of competence for DNA synthesis in the wing discs. Both DNA synthesis and protein content were drastically reduced in plumbagin and azadirachtin-treated insects. Underin vitro conditions, plumbagin had a more pronounced inhibitory effect than azadirachtin. All the ecdysteroids tested, viz. makisterone A, 20-hydroxyecdysone and the ecdysteroidal fraction from the silver fernCheilanthes farinosa enhanced DNA synthesis     

Chromosome Localization of the λ20p1.4 clone of the Drosophila melanogaster Nuclear Lamina DNA in the melanogaster Species Subgroup of the Genus Drosophila (Sophophora)

Chromosome localization of sequences homologous to 20p1.4 of the Drosophila melanogaster nuclear lamina DNA (nlDNA) was established by in situ hybridization in species of the melanogastersubgroup. DNA of the 20p1.4 clone was shown to be located in the chromocenter in all the species examined. Laboratory strains of D. simulans, D. mauritiana, andD. sechellia exhibited interspecific differences in localization of 20p1.4 nlDNA on chromosome arms. In eight natural populations, intraspecific polymorphism of 20p1.4 nlDNA chromosome localization was shown to be present in D. simulans but absent in D. melanogaster. The possible participation of transposable elements in 20p1.4 nlDNA relocation is discussed.     

Organization of the lamina ganglionaris of the optic lobe of the butterfly Pararge aegeria (Linné) (Lepidoptera : Satyridae)

The present work reports on a neuroanatomical study of the butterfly Pararge aegeria (Lepidoptera : Satyridae) focusing on the lamina ganglionaris underlying two different regions of the retina of the compound eye: the dorsal rim area and the large dorsal region. No differences between both lamina regions, concerning the structure of the cartridges and the morphology of the identified neurons, could be detected. After passing the basement membrane, the visual cell axons are organized in retinotopic bundles (pseudocartridges), in which the axons of the 9 visual cells (V1 and 5, D2, 4, 6, 8, H3 and 7, B9) are arranged in the same way as in the retina. In the pseudocartridge there are no synaptic contacts. Before entering the lamina cartridge, the bundles rotate 90 °. The cartridges are joined by the fibres of 4 monopolar cells (L1, L2, L3 and L4), which could be identified and located inside the lamina cartridges in serial EM-sections. Golgi impregnations revealed the morphology of these fibres. Thus, the regional specialization of the retina (dorsal rim area and large dorsal region) does not seem to be reflected at the level of the first visual neuropil. Additionally, the cartridges of both lamina regions were investigated qualitatively for synaptic contacts among fibres. In addition to monadic chemical synapses and multiple contact synapses with presynaptic ribbons, cell contacts are also facilitated by invaginations and bridges. These cellular interactions and their functional implications are discussed.     

DNA–DNA Hybridization Evidence for the Recent Origin of Marmots and Ground Squirrels (Rodentia: Sciuridae)

A molecular analysis, based on DNA/DNA hybridization experiments, examined seven species of the sciurid tribe Marmotini, in order to evaluate their evolutionary relationships. The branching pattern obtained from a neighbor-joining analysis and least-squares methods indicates that spermophiles and marmots are closely related. This result is in agreement with previous studies based on biochemical data which have suggested a Miospermophilus origin of Marmota (like Spermophilus), instead of the Protospermophilus origin usually proposed by paleontology. Using a molecular time scale calibrated by the fossil record of Sciurus (used here as an outgroup), we estimate the marmot/ground squirrel divergence at 6 My, at the same time as the major lineages of ground squirrels diverged from each other. Consequently, the species of Marmota appear as one of the numerous specialized forms derived from the explosive radiation of the Spermophilus lineage in the late Miocene.     

Phylogeny of the Alismatales (Monocotyledons) and the relationship of Acorus (Acorales?)

A phylogenetic analysis of the early branching lineages of the monocotyledons is performed using data from two plastid genes (rbcL and matK), five mitochondrial genes (atp1, ccmB, cob, mttB and nad5) and morphology. The complete matrix includes 93 terminals representing Acorus, the 14 families currently recognized within Alismatales, and numerous lineages of monocotyledons and other angiosperms. Total evidence analysis results in an almost completely resolved strict consensus tree, but all data partitions, genomic as well as morphological, are incongruent. The effects of RNA editing and potentially processed paralogous sequences are explored and discussed. Despite a decrease in incongruence length differences after exclusion of edited sites, the major data partitions remain significantly incongruent. The 14 families of Alismatales are all found to be monophyletic, but Acorus is found to be included in Alismatales rather than being the sister group to all other monocotyledons. The placement is strongly supported by the mitochondrial data, atp1 in particular, but it cannot be explained as an artifact caused by patterns of editing or by sampling of processed paralogues.     

DNA barcoding in native plants of the Labiatae (Lamiaceae) family from Chios Island (Greece) and the adjacent Çeşme-Karaburun Peninsula (Turkey)

The plant family Labiatae (Lamiaceae) is known for its fine medicinal and aromatic herbs like lavender, mint, oregano, sage and thyme and is a rich source of essential oils for the food, pharmaceutical and cosmetic industry. Besides its great economic importance, the Labiatae family contributes significantly to the endemic flora of Greece and Turkey. Owing to its economic and biological significance and to the difficult identification based on morphological characters of several of its taxa, the Labiatae family is an ideal case for developing DNA barcodes. The purpose of this study is to evaluate the utility of DNA barcoding on a local scale in discriminating Labiatae species in Chios Island (Greece) and the adjacent Çe?me‐Karaburun Peninsula (Turkey). We chose three cpDNA regions (matK, rbcL, trnH‐psbA) that were proposed by previous studies and tested them either as single region or as multiregion barcodes based on the criteria determined by Consortium for the Barcode of Life (CBOL). Our results show that matK and trnH‐psbA taken as useful in discriminating species of the Labiatae, for the species we examined, as any multiregion combination. matK and trnH‐psbA could serve as single‐region barcodes for Labiatae species contributing to the conservation and the trade control of valuable plant resources.     

Effect of the alkaline cations on the stability of the model polynucleotide poly(dG-dC)·poly(dG-dC)

When the model polynucleotide poly(dG-dC)?poly(dG-dC) [polyGC] is titrated with a strong acid (HCl) in unbuffered aqueous solutions containing the chlorides of the alkali metals in the concentration range 0.010?M-0.600?M, two transitions in the absorbance vs. pH plots are evidenced, characterized by the constants pK(a(?)) and pK(a(?)). The limiting values at infinite saline concentrations of these two constants, namely pK(∞)(a(?)) and pK(∞)(a(?)) obtained making use of the "one site saturation constant" equation or, in turn, of the double logarithmic plot: pK(a) vs. log([salt]?1), exhibit a clear dependence on the nature of the cations. The effects of the different alkali cations on the pK(∞)(a) values follow the Hofmeister series. In fact, the pK(∞)(a(?)) and the pK(∞)(a(?)) values are smaller for Li+ and Na+ than for Rb+ and Cs+, with K+ at the border between the two, showing that the transitions require higher concentrations of protons to occur in the presence of high concentrations of the cosmotropic ions.     

Recombinant DNA cloning of the active region of the receptor activator of NF-κB ligand (RANKL) gene and its role in osteoclastogenesis

Osteopetrosis belongs to a group of rare genetic diseases typically treated with bone marrow transplantation. This approach is not effective in a recently identified form of the disease caused by mutations in the receptor activator of NF-κB ligand (RANKL) gene. In these patients, replacement therapy and RANKL delivery may be a more valid approach than transplantation. Here, we describe the construction of a recombinant gene encoding regions of RANKL (rRANKL), including the biologically active regional loop sequence. We present detailed methods for the cloning, expression, and purification of the recombinant protein. The activity of rRANKL including the active region was assessed in vitro and mature osteoclast generation was evaluated in vivo using a mouse model. We provide a proof of concept for the therapeutic potential of full-length and selected active regions of rRANKL in the treatment of osteopetrosis, warranting clinical assessment.     

Diminution and re-synthesis of DNA during development and senescence of the “diploid” macronuclei of the ciliate Trachelonema sulcata (Gymnostomata,Karyorelictida)

The DNA content of micronuclei, macronuclear anlagen, and of macronuclei of three age categories (young, mature and old) has been studied by cytophotometry of Feulgen stained isolated nuclei. The DNA content of the macronuclear anlagen decreases, at average, to one half of that of the micronuclei, from which they develop. Accumulations of fibro-granular material have been revealed electron microscopically at the periphery of such anlagen (in the thin perinuclear layer of cytoplasm); this material seems to have a nuclear origin. The DNA content of young macronuclei remains as low as that of the anlagen, but in mature ones it again increases approximately to the level of the micronuclei. The DNA content of old macronuclei is highly variable and ranges from equal to that of a micronucleus to exceeding this level about six times. These data indicate that a part of the genome of the micronuclei is lost at the beginning of their transformation into macronuclei, and that there is subsequent partial replication of some DNA fractions in maturing and ageing macronuclei.     

Characterization of the single-stranded DNA binding protein pV(VGJΦ) of VGJΦ phage from Vibrio cholerae

pV(VGJΦ), a single-stranded DNA binding protein of the vibriophage VGJΦ was subject to biochemical analysis. Here, we show that this protein has a general affinity for single-stranded DNA (ssDNA) as documented by Electrophoretic Mobility Shift Assay (EMSA). The apparent molecular weight of the monomer is about 12.7kDa as measured by HPLC-SEC. Moreover, isoelectrofocusing showed an isoelectric point for pV(VGJΦ) of 6.82 pH units. Size exclusion chromatography in 150mM NaCl, 50mM sodium phosphate buffer, pH 7.0 revealed a major protein species of 27.0kDa, suggesting homodimeric protein architecture. Furthermore, pV(VGJΦ) binds ssDNA at extreme temperatures and the complex was stable after extended incubation times. Upon frozen storage at -20°C for a year the protein retained its integrity, biological activity and oligomericity. On the other hand, bioinformatics analysis predicted that pV(VGJΦ) protein has a disordered C-terminal, which might be involved in its functional activity. All the aforementioned features make pV(VGJΦ) interesting for biotechnological applications.     

Exploration of the binding mode of α/β-type small acid soluble proteins (SASPs) with DNA

The α/β-type small acid soluble proteins (SASPs) are a major factor in protecting the spores from being killed in bacteria. In this article, we perform a systematic phylogenetic analysis of the α/β-type SASP in the genus of Geobacillus, which indicates that the whole family can be divided into three groups. We choose one protein from each group as a representative and construct the tertiary structure of these proteins. In order to explore the mechanism of protecting DNA from damage, 15 ns molecular dynamics simulation for the four complexes of Gsy3 with DNA are performed. The sequence alignment, model structure and binding energy analysis indicate that the helix2 region of SASPs is more conserved and plays a more crucial role in protecting DNA. Pairwise decomposition of residue interaction energies calculation demonstrate that amino acids of Asn10, Lys24, Asn49, Ile52, Ile56, Thr57, Lys58, Arg59 and Val61 take major effect in the binding interaction. The differences of energy contribution of the amino acids between different complexes make us conclude that the protein structure conformation has a slight change upon more proteins binding to DNA and consequently there occur protein-protein cooperation interactions.     

Mitochondrial Single-stranded DNA-binding Proteins Stimulate the Activity of DNA Polymerase γ by Organization of the Template DNA

The activity of the mitochondrial replicase, DNA polymerase γ (Pol γ) is stimulated by another key component of the mitochondrial replisome, the mitochondrial single-stranded DNA-binding protein (mtSSB). We have performed a comparative analysis of the human and Drosophila Pols γ with their cognate mtSSBs, evaluating their functional relationships using a combined approach of biochemical assays and electron microscopy. We found that increasing concentrations of both mtSSBs led to the elimination of template secondary structure and gradual opening of the template DNA, through a series of visually similar template species. The stimulatory effect of mtSSB on Pol γ on these ssDNA templates is not species-specific. We observed that human mtSSB can be substituted by its Drosophila homologue, and vice versa, finding that a lower concentration of insect mtSSB promotes efficient stimulation of either Pol. Notably, distinct phases of the stimulation by both mtSSBs are distinguishable, and they are characterized by a similar organization of the template DNA for both Pols γ. We conclude that organization of the template DNA is the major factor contributing to the stimulation of Pol γ activity. Additionally, we observed that human Pol γ preferentially utilizes compacted templates, whereas the insect enzyme achieves its maximal activity on open templates, emphasizing the relative importance of template DNA organization in modulating Pol γ activity and the variation among systems.     

Structure of the RNA claw of the DNA packaging motor of bacteriophage ?29

Bacteriophage DNA packaging motors translocate their genomic DNA into viral heads, compacting it to near-crystalline density. The Bacillus subtilis phage ϕ29 has a unique ring of RNA (pRNA) that is an essential component of its motor, serving as a scaffold for the packaging ATPase. Previously, deletion of a three-base bulge (18-CCA-20) in the pRNA A-helix was shown to abolish packaging activity. Here, we solved the structure of this crucial bulge by nuclear magnetic resonance (NMR) using a 27mer RNA fragment containing the bulge (27b). The bulge actually involves five nucleotides (17-UCCA-20 and A100), as U17 and A100 are not base paired as predicted. Mutational analysis showed these newly identified bulge residues are important for DNA packaging. The bulge introduces a 33–35° bend in the helical axis, and inter-helical motion around this bend appears to be restricted. A model of the functional 120b pRNA was generated using a 27b NMR structure and the crystal structure of the 66b prohead-binding domain. Fitting this model into a cryo-EM map generated a pentameric pRNA structure; five helices projecting from the pRNA ring resemble an RNA claw. Biochemical analysis suggested that this shape is important for coordinated motor action required for DNA translocation.