Binding of adenosine diphosphoribosyltransferase to the termini and internal regions of linear DNAs

Binding mechanisms of ADPR-transferase to restricted double-stranded DNA fragments of SV40 and pBR322 DNA were determined by nuclease protection techniques. Top and bottom strands of double-stranded DNA were identified by specific labeling with 32P. Protection against specific exonucleases identified binding of ADPR-transferase to DNA termini, whereas binding to internal regions of linear DNAs was probed by protection against endonucleases. ADPR-transferase protein protected against exonucleolytic attack from lambda exo and exoIII in all DNA fragments tested, demonstrating that the enzyme protein binds indiscriminately to all DNA termini. Extending earlier results [Sastry, S.S., & Kun, E. (1988) J. Biol. Chem. 263, 1505-1512], the modifying effect of the binding of ADPR-transferase to DNA induced changes in DNA conformation, as evident from altered pause sites that appeared following digestion of DNA fragments by lambda exonuclease in the presence of ADPR-transferase. In contrast to the nonselective binding of ADPR-transferase to DNA termini, ADPR-transferase conferred protection endonuclease attack (DNase I and micrococcal nuclease) only to the 209-bp EcoRI-PstI SV40 DNA fragment. These results indicate that binding of ADPR-transferase to relatively rare internal regions of restricted DNA fragments exhibits some degree of specificity. Specificity of binding appears to be related to the coincidental relative A+T-rich regions in DNA, and to DNA bending, both identified in the 209-bp SV40 DNA fragment. Synthetic polydeoxyribonucleotides containing dA-dT bind ADPR-transferase stronger than polydeoxyribonucleotides containing dG-dC. It was deduced from endonuclease protection patterns that binding of the enzyme protein leaves no defined footprints on the 209-bp SV40 DNA fragment, but there is significant modification of DNA structure following binding of the enzyme protein. Methylation protection assays and the prevention of the binding of ADPR-transferase to T4 DNA by its glucosylation indicate that the enzyme binds in the major groove of DNA. The 36-kDa A peptide fragment of ADPR-transferase [Buki, K. G., & Kun, E. (1988) Biochemistry 27, 5990-5995] exhibits the same protection against endonucleolytic enzymes as the intact ADPR-transferase molecule.     

Single-stranded regions in Streptococcus pneumoniae chromosomal deoxyribonucleic acid and their relation to transformation.

Deoxyribonucleic acid (DNA) in lysates of both completent and noncompetent streptococcus pneumoniae cells was characterized by chromatography on benzoylated, naphthoylated diethylaminoethyl-cellulose columns, by sensitivity to Aspergillus oryzae S1 endonuclease, and by sucrose gradient analysis. The DNAs from both competent and noncompetent cells were found to contain similar extents of single-stranded regions. These single-stranded regions appeared to be intact, unpaired regions in double-stranded DNA rather than gaps, nicks, or unpaired ends in the DNA. Inhibition of cells with rifampin prior to lysis increased the amount of such single strandedness in the DNA. Lysates made at various times after [14C]thymidine-labeled cells had bound [3H]thymidine-labeled transforming DNA were also characterized by benzoylated, naphthoylated diethylaminoethyl-cellulose chromatography. Changes in the elution profiles of DNA from cells exposed to homospecific (S. pneumoniae) donor DNA were indicative of the formation of complexes between donor DNA and the single-stranded regions of recipient DNA. In contrast, profiles of DNA from cells exposed to heterospecific (S. sanguis) DNA did not show significant changes, indicating that few such donor-recipient complexes were formed during heterospecific transformation.     

Different functional sites on rIFN-alpha 2 and their relation to the cellular receptor binding site

Functional domains on the recombinant interferon-alpha 2 (rIFN-alpha 2) molecule, which are involved in antiviral and NK enhancing activities, have been defined by immunochemical mapping with MAb, and their relationship with the IFN cellular receptor binding site has been studied. With 20 different anti-IFN-alpha 2 MAb selected by their binding to 125I-labeled IFN and by immunoprecipitation of the 20 Kd IFN molecule, we have defined three spatially separated epitopes (designated as sites A, B, and C) and two partially overlapping antigenic determinants on the IFN-alpha 2 molecule. Functional relation of IFN-alpha 2 A, B, and C epitopes have been determined by assaying the effect of various anti-IFN MAb on IFN-mediated biologic activities. MAb directed to sites A and B neutralized the antiviral activity of IFN. Furthermore, the MAb specific for site B displayed a neutralizing potency threefold higher than MAb directed to site A. Site B was also involved in the enhancing activity of IFN on NK-mediated cell cytotoxicity, whereas site A was not. MAb directed to site C partially affected the IFN-boosted NK activity but did not neutralize the IFN antiviral activity. Inhibition studies of 125I-IFN binding to human U-937 myelomonocytic cells by anti-IFN MAb demonstrated that MAb directed to site B blocked different IFN biologic functions by preventing its binding to the cellular receptor, whereas MAb directed to sites A and C caused no inhibition and partial inhibition of this binding, respectively.     

RFLP-based mapping of three mutant loci in rye (Secale cereale L.) and their relation to homoeologous loci within the Gramineae

 Three mutant loci of rye determining absence of ligules (al), waxless plant (wa1) and waxy endosperm (Wx) characters were mapped in a single F₂ population, comprising 84 individual plants. The three loci could be clearly tagged in relation to 7 (al on chromosome 2R), 4 (wa1 on chromosome 7R) or 6 (Wx on chromosome 4R) RFLP markers. The mapping data are compared with existing data for homoeologous regions containing equivalent mutants of wheat, barley, rice and maize. It is shown that the loci analysed are highly conserved across the cereal species, including rye. Received: 14 March 1997 / Accepted: 21 March 1997     

Dynamics in natural and designed elastins and their relation to elastic fiber structure and recoil

Elastin fibers assemble in the extracellular matrix from the precursor protein tropoelastin and provide the flexibility and spontaneous recoil required for arterial function. Unlike many proteins, a structure-function mechanism for elastin has been elusive. We have performed detailed NMR relaxation studies of the dynamics of the minielastins 24x′ and 20x′ using solution NMR, and of purified bovine elastin fibers in the presence and absence of mechanical stress using solid state NMR. The low sequence complexity of the minielastins enables us to determine average dynamical timescales and degrees of local ordering in the cross-link and hydrophobic modules separately using NMR relaxation by taking advantage of their residue-specific resolution. We find an extremely high degree of disorder, with order parameters for the entirety of the hydrophobic domains near zero, resembling that of simple chemical polymers and less than the order parameters that have been observed in other intrinsically disordered proteins. We find that average backbone order parameters in natural, purified elastin fibers are comparable to those found in 24x′ and 20x′ in solution. The difference in dynamics, compared with the minielastins, is that backbone correlation times are significantly slowed in purified elastin. Moreover, when elastin is mechanically stretched, the high chain disorder in purified elastin is retained, showing that any change in local ordering is below that detectable in our experiment. Combined with our previous finding of a 10-fold increase in the ordering of water when fully hydrated elastin fibers are stretched by 50%, these results support the hypothesis that stretch induced solvent ordering, i.e., the hydrophobic effect, is a key player in the elastic recoil of elastin as opposed to configurational entropy loss.     

Structure of the mouth opening and pharyngeal apparatus in three species of Baikal Cottoidei in relation to their feeding

The structure of the mouth opening and pharyngeal apparatus was studied, and its relation to the feeding pattern in three coastal species of Baikal Cottidae fish was analyzed. Analysis of data demonstrated that the general pattern of structure of the mouth opening and pharyngeal apparatus is similar in the species under study since their food is represented by bottom organisms, mainly by amphipods. Interspecies differences are manifested in an increase in relative values of morphological features and the size of consumed prey.     

Peripheral regions of natural hammerhead ribozymes greatly increase their self-cleavage activity

Natural hammerhead ribozymes are mostly found in some viroid and viroid-like RNAs and catalyze their cis cleavage during replication. Hammerheads have been manipulated to act in trans and assumed to have a similar catalytic behavior in this artificial context. However, we show here that two natural cis-acting hammerheads self-cleave much faster than trans-acting derivatives and other reported artificial hammerheads. Moreover, modifications of the peripheral loops 1 and 2 of one of these natural hammerheads induced a >100-fold reduction of the self-cleavage constant, whereas engineering a trans-acting artificial hammerhead into a cis derivative by introducing a loop 1 had no effect. These data show that regions external to the central conserved core of natural hammerheads play a role in catalysis, and suggest the existence of tertiary interactions between these peripheral regions. The interactions, determined by the sequence and size of loops 1 and 2 and most likely of helices I and II, must result from natural selection and should be studied in order to better understand the hammerhead requirements in vivo.     

Monitoring ecological indicators of rangeland functional integrity and their relation to biodiversity at local to regional scales

Abstract Functional integrity is the intactness of soil and native vegetation patterns and the processes that maintain these patterns. In Australia's rangelands, the integrity of these patterns and processes have been modified by clearing, grazing and fire. Intuitively, biodiversity should be strongly related to functional integrity; that is, landscapes with high functional integrity should maintain biodiversity, and altered, less functional landscapes may lose some biodiversity, defined here as the variety and abundance of the plants, animals and microorganisms of concern. Simple indicators of biodiversity and functional integrity are needed that can be monitored at a range of scales, from fine to coarse. In the present paper, we use examples, primarily from published work on Australia's rangeland, to document that at finer patch and hillslope scales several indicators of landscape functional integrity have been identified. These indicators, based on the quantity and quality of vegetation patches and interpatch zones, are related to biodiversity. For example, a decrease in the cover and width (quantity) and condition (quality) of vegetation patches, and an increase in bare soil (quantity of interpatch) near cattle watering points in a paddock are significantly related to declines in plant and grasshopper diversity. These vegetation patch‐cover and bare‐soil indicators have been monitored traditionally by field‐based methods, but new high‐resolution, remote‐sensing imagery can be used in specific rangeland areas for this fine‐scale monitoring. At intermediate paddock and small watershed scales, indicators that can be derived from medium‐resolution remote‐sensing are also needed for efficient monitoring of rangeland condition (i.e. functional integrity) and biodiversity. For example, 30–100‐m‐pixel Landsat imagery has been used to assess the condition of rangelands along grazing gradients extending from watering‐points. The variety and abundance of key taxa have been related to these gradients (the Biograze project). At still larger region and catchment scales, indicators of rangeland functional integrity can also be monitored by coarse‐resolution remote‐sensing and related to biodiversity. For example, the extent and greenness (condition) of different regional landscapes have been monitored with 1‐km‐pixel satellite imagery. This regional information becomes more valuable when it indicates differences as a result of land management. Finally, we discuss potential future developments that could improve proposed indicators of landscape functional integrity and biodiversity, thereby improving our ability to monitor rangelands effectively.     

Structural and functional characteristics of epiphyton and epipelon in relation to their distribution in Lake Vechten

Epiphyton and epipelon were quantitatively collected, respectively, from the submerged macrophytes and the sandy lake bottom of Lake Vechten (The Netherlands). On a weight basis, epiphyton was maximal in autumn and epipelon in summer. In winter the chemical composition of epiphyton and epipelon was similar. In summer the epiphyton had on a unit weight basis more organic matter and carbonate, and had per unit organic matter a higher algal number, nitrogen and energy content than the epipelon. Algae predominating the epiphyton were filamentous greens and pennate diatoms; those in the epipelon were pennate diatoms and blue-green algae. In both cases, species known to frequent the phytoplankton were abundant. The diatoms were quantified using paper chromatographic pigment analyses. Both the epiphyton and the epipelon exhibited maximal photosynthesis in mid summer. That light was generally the limiting factor was evident from periphyton developed on artificial substrates. This periphyton differed widely in its composition from that on the natural substrates, mainly because the latter collected much more sedimenting matter.In dense Ceratophyllum stands light was severely attenuated and the significant gradients in oxygen and pH were caused by the differences with depth in the proportions of photosynthesis and respiration. The oxygen content and pH at the bottom decreased owing to epipelic respiration. The epiphytic composition depended greatly on the degree of light attenuation. The epiphytic and epipelic respiration, except during part of the early summer, exceeded photosynthesis on a 24 h basis; this included the macrophytic photosynthesis during the time the vegetation was maximally developed. During the growing season import of organic matter, i.e. deposited seston, greatly exceeded that due to the photosynthetic production. After the summer maximum, the epipelon decreased faster than predicted from its oxygen exchange. It was concluded that sedimentation and resuspension determined mainly the changes in epiphyton and epipelon. Especially when covered with vegetation, the lower littoral of Lake Vechten plays a large part in the aerobic decomposition of sestonic organic matter.     

Pressurized gas transport in two Japanese alder species in relation to their natural habitats

Oxygen uptake measurements have shown that pressurized gas transport, resulting from the physical effect of thermo-osmosis of gases, improves oxygen supply to the roots of the seedlings in two alder speciesAlnus japonica (Thunb.) Steud. andAlnus hirsuta (Spach) Rupr., which are both native in Japan. When gas transport conditions were established by irradiation of the tree stems the internal aeration was increased to a level nearly equal to the oxygen demand of the root system in leafless seedlings ofA. hirsuta, but was higher inA. japonica so that excess oxygen was excreted into the environment. An increase of superoxide dismutase (SOD) activity, which protects plants from toxic oxygen radicals and post-anoxic injury, has been observed in root tissues ofA. japonica when the seedlings were flooded for 3 days. The increase of SOD activity, in concert with high gas transport rates, may enable this tree species to grow in wet sites characterized by low oxygen partial pressure in the soil and by varying water tables. A less effective gas transport, flood-induced reduction of SOD activity in root tissues, and reduced height growth in waterlogged soil may be responsible for the fact thatA. hirsuta is unable to inhabit wettland sites.     

Characteristics ofBacteroides fragilis bacteriophages and comparison of their DNAs

Although fiveBacteroides fragilis bacteriophages isolated over a six-year period in Nebraska and Virginia had similar physical characteristics (morphology, temperature inactivation, and sensitivity to organic solvents and antisera), there were some statistically significant differences between the phages. In addition, restriction endonuclease analysis revealed that three of the five DNAs were not identical. However, the DNAs of the phages were closely related based on DNA-DNA hybridization, percent homologies, and possession of homologous regions of DNA. It appears that the five phages are strains of the same species of phage, although each phage has a unique host range spectrum.     

The ecology of hungry larvae of Dermacentor reticulatus and their relation to food hosts under natural conditions

Peculiarities of activation and regularities of spatial and temporary spread of hungry larvae of D. reticulatus from the progeny of one female were studied. Activation of larvae occurs within 16 to 20 days. Under natural conditions the activation of approximately 5 to 15% of potential number of individuals produced by a single clutch takes place. A larval aggregation occupies a territory of several square metres and survives about a month. Larvae from two and more clutches feed simultaneously on small mammals. Individual larvae from the progeny of one female usually feed on one animal. The spread of larvae by the hosts occurs apparently within the limits of some hectares.     

Locations of mouse DNA damage response and repair loci, and cancer risk modifiers

An outline is presented of an electronically accessible database that compares the locations of mouse genes involved in DNA repair, apoptosis, cell cycle and signal transduction with those of known cancer risk modifier genes. The database has a primary but not exclusive focus on modifiers of ionizing radiation (IR) cancer risk and genes involved in IR-induced DNA damage responses. The database () provides a useful tool for assessing the role of DNA damage response genes in cancer predisposition.     

A novel approach to segregate and identify functional loop regions in protein structures using their Ramachandran maps

The loops which connect or flank helices/sheets in protein structures are known to be functionally important. However, ironically they also belong to the part of protein whose structure is least accurately predicted. Here, a new method to isolate and analyze loop regions in protein structure is proposed using the spatial coordinates of the solved three‐dimensional structure. The extent of dispersion among points of successive amino acid residues in the Ramachandran map of protein region is utilized to calculate the Mean Separation between these points in the Ramachandran Plot (MSRP). Based on analysis of 2935 protein secondary structure regions obtained using DSSP software, spanning a range from 2 to 64 residues, taken from a set of 170 proteins, it is shown that helices (MSRP < 17) and strands (MSRP < 64) stand effectively demarcated from the loop regions (MSRP > 130). Analysis of 43 DNA binding and 98 ligand binding proteins revealed several loop regions with clear change in MSRP subsequent to binding. The population of such loops correlated with the magnitude of backbone displacement in the protein subsequent to binding. Can changes in MSRP quantify the temporal oscillations in dihedral angles among structured/unstructured regions in proteins? Molecular dynamics simulations (10 ns) revealed that deviations in MSRP among different snapshots in the trajectory were at least twofold higher for unstructured proteins in comparison with ordered proteins. The above results validate the use of MSRP parameter as a tool to identify and investigate functionally active loops and unstructured regions in protein structures. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Structure of extrachromosomal circular DNAs excised from T-cell antigen receptor alpha and delta-chain loci

Small polydisperse circular (spc) DNA was isolated from mouse thymocytes, fragmented by HindIII digestion and cloned into the vector. Sixty DNA clones were randomly selected from the 10,400 phage library. The average size of insert was one-fifth of the original circular molecule. Twenty spc-DNA clones were homologous to DNA probes derived from T-cell antigen receptor (TCR) alpha-chain loci. We have characterized nine clones by DNA sequencing; they contain new germline sequences of the TCR alpha-chain variable (V alpha) and joining (J alpha) gene segments and the products out of the recombination of a V alpha with a J alpha gene segment. An additional four spc-DNA clones carried a new rearranging gene of the TCR delta-chain that is located between V alpha and J alpha genes. At least nine of 60 DNA clones carried the recombination junction of a heptamer-heptamer head-to-head structure expected from an excised product of V-J joining. This shows that most extrachromosomal circular DNAs in the thymus are formed by a sequence-dependent recombination mechanism. We suggest that a functional T-cell receptor V alpha gene can be constructed by somatic random rearrangements through successive looping-out, excision and deletion.     

Locations and contexts of sequences that hybridize to poly(dG-dT).(dC-dA) in mammalian ribosomal DNAs and two X-linked genes.

Sequences located several kilobases both 5' and 3' of the stably transcribed portion of several genes hybridize to radio-labeled pure fragments of the alternating sequence poly (dG-dT) (dC-dA) ["poly(GT)"]. The genes include the ribosomal DNA of mouse, rat, and human, and also human glucose-6-phosphate dehydrogenase (G6PD) and mouse hypoxanthine-guanine phosphoribosyl transferase (HPRT). HPRT has additional hybridizing sequences in introns. Fragments that include the hybridizing sequences and up to 300 bp of adjoining DNA show perfect runs of poly(GT) (greater than 30bp) in all but the human 5' region of rDNA, which shows a somewhat different alternating purine:pyrimidine sequence, poly(GTAT) (36bp). Within 150 bp of these sequences in various instances are found a number of other sequences reported to affect DNA conformation in model systems. Most marked is an enhancement of sequences matching at least 67% to the consensus binding sequence for topoisomerase II. Two to ten-fold less of such sequences were found in other sequenced portions of the nontranscribed spacer or in the transcribed portion of rDNA. The conservation of the locations of tracts of alternating purine:pyrimidine between evolutionarily diverse species is consistent with a possible functional role for these sequences.