Dynamic DNA contacts observed in the NMR structure of winged helix protein-DNA complex.

Genesis is an HNF-3/fkh homologous protein. By using multi-dimensional NMR techniques, we have obtained the solution structure and backbone dynamics of Genesis complexed with a 17 base-pair DNA. Our results indicate that both the local folding and dynamic properties of Genesis are perturbed when it binds to the DNA site. Our data show that a conserved flexible amino acid sequence (wing 1) makes dynamic contacts to DNA in the complex and a short helix is induced by Genesis-DNA interactions. Our data indicate that, unlike the HNF-3gamma/DNA complex, a magnesium ion is not required in forming the stable Genesis-DNA complex.     

Inhibition of apoptosis by zinc: a reappraisal.

Apoptosis--or programmed cell death--is an active type of cell death, occurring in several pathophysiological conditions. One of the most important characteristics of apoptosis is that cell death is preceded by DNA fragmentation, consequent to the activation of nuclear calcium- and magnesium-dependent endonuclease(s). DNA fragmentation can be inhibited by zinc ions. By using several techniques, such as DNA agarose gel electrophoresis, cytofluorimetric analysis of DNA content and of cell cycle, 3H-thymidine incorporation and trypan blue dye exclusion test, we show that zinc, despite completely inhibiting DNA fragmentation and the consequent loss of nuclear DNA content, does not protect rat thymocytes from spontaneous or dexamethasone-induced death. Our data also suggest that DNA fragmentation, although characteristic, is not a critical event for thymocyte death of apoptotic type.     

Nucleosome arrangement in alpha-satellite chromatin of African green monkey cells.

By analyzing the accessibility of restriction endonuclease sites in African green monkey alpha-satellite chromatin, we demonstrate the absence of a unique phase relationship between nucleosomes and alpha-satellite DNA. The data indicate a minimum of three different positions for nucleosome cores relative to the alpha-satellite sequence and suggest a random distribution in at least some regions. In addition, while we confirm published reports that staphylococcal nuclease cuts the alpha-satellite sequence in chromatin at a highly preferred site, two-dimensional gel electrophoresis of nuclear digests demonstrates that this site is preferentially cut by staphylococcal nuclease even when it is within the nucleosome core. These data indicate that staphylococcal nuclease is not useful for determining nucleosome positions on alpha-satellite DNA, and perhaps on other specific DNA sequences as well.     

Express yourself: bold individuals induce enhanced morphological defences

Organisms display an impressive array of defence strategies in nature. Inducible defences (changes in morphology and/or behaviour within a prey''s lifetime) allow prey to decrease vulnerability to predators and avoid unnecessary costs of expression. Many studies report considerable interindividual variation in the degree to which inducible defences are expressed, yet what underlies this variation is poorly understood. Here, we show that individuals differing in a key personality trait also differ in the magnitude of morphological defence expression. Crucian carp showing risky behaviours (bold individuals) expressed a significantly greater morphological defence response when exposed to a natural enemy when compared with shy individuals. Furthermore, we show that fish of different personality types differ in their behavioural plasticity, with shy fish exhibiting greater absolute plasticity than bold fish. Our data suggest that individuals with bold personalities may be able to compensate for their risk-prone behavioural type by expressing enhanced morphological defences.     

Consistency in Context-specific Measures of Shyness and Boldness in Rainbow Trout, Oncorhynchus mykiss

Shyness and boldness has been considered a fundamental axis of human behavioural variation. At the extreme ends of this behavioural continuum subjects vary from being bold and assertive to shy and timid. Analogous patterns of individual variation have been noted in a number of species including fish. There has been debate on the nature of this continuum as to whether it depends on context. That is, whether it is domain‐general (as in humans), or context‐specific. The purpose of our study was to test if shyness and boldness depends on context in rainbow trout, Oncorhynchus mykiss and to this end we estimated boldness in five different situations. Our data provide evidence of a shy–bold behavioural syndrome in rainbow trout. Bold trout tended to be bold in four situations when the context was similar (when the context concerned foraging). However, in a different context, exploring a swim flume, the ranking was entirely different. We suggest that shyness and boldness depends on context in rainbow trout.     

Ancient DNA from pollen: a genetic record of population history in Scots pine

Assessments of plant population dynamics in space and time have depended on dated records of fossil pollen synthesized on a subcontinental scale. Genetic analyses of extant populations have revealed spatial relationships that are indicative of past spatial dynamics, but lack an explicit timescale. Synthesis of these data requires genetic analyses from abundant dated fossil material, and this has hitherto been lacking. Fossil pollen is the most abundant material with which to fill this data gap. Here we report genetic analyses of fossil pollen retrieved from Holtjärnen postglacial lake sediment in Sweden and show that plastid DNA is recoverable from Scots Pine and Norway spruce pollen grains that are 100 and 10 000 years old. By sequencing clones from two short plastid PCR products and by using multiple controls we show that the ancient sequences were endogenous to the fossil grains. Comparison of ancient sequences and those obtained from an extant population of Scots pine establishes the first genetic link between extant and fossil samples in this species, providing genetic continuity through time. The finding of one common haplotype present in modern, 100-year old and 10 000-year old samples suggests that it may have persisted near Holtjärnen throughout the postglacial period. This retrieval of ancient DNA from pollen has major implications for plant palaeoecology in conifer species by allowing direct estimates of population dynamics in space and time.     

Significance of dimers to the size of newly synthesized DNA in UV-irradiated Chinese hamster ovary cells.

DNA synthesized after UV irradiation is smaller than that in unirradiated cells even when pulse-labeling times are increased to compensate for the overall reduction in the rate of DNA replication. By isolating newly replicated DNA, incubating it with dimer-specific endonuclease from Micrococcus luteus, and analyzing it on alkaline sucrose gradients, we have been able to demonstrate that this DNA is synthesized in segments corresponding in size to the interdimer distance on the parental strand. In addition, the same DNA analyzed on neutral gradients shows no reduction in molecular weight as a result of UV irradiation and/or endonuclease digestion. Our data are thus inconsistent with the presence of "gaps" in newly synthesized DNA opposite the dimers on the parental strand. We suggest that if such gaps are produced as a result of delayed synthesis around dimers, they are filled before the growing point reaches the next dimer.     

Shoal composition determines foraging success in the guppy

The composition of an animal group can impact greatly on thesurvival and success of its individual members. Much recentwork has concentrated on behavioral variation within animalpopulations along the bold/shy continuum. Here, we screenedindividual guppies, Poecilia reticulata, for boldness usingan overhead fright stimulus. We created groups consisting of4 bold individuals (bold shoals), 4 shy individuals (shy shoals),or 2 bold and 2 shy individuals (mixed shoals). The performanceof these different shoal types was then tested in a novel foragingscenario. We found that both bold and mixed shoals approacheda novel feeder in less time than shy shoals. Interestingly,we found that more fish from mixed shoals fed than in eitherbold or shy shoals. We suggest that this can be explained bythe fact that nearly all the cases where one fish was followedinto the feeder by another occurred within mixed shoals andthat it was almost always a shy fish following a bold one. Theseresults suggest clear foraging benefits to shy individuals throughassociating with bold ones. Surprisingly, our results also suggestpotential foraging benefits to bold individuals through associatingwith shy individuals. This study highlights a possible mechanismby which interindividual variation in behavioral types is maintainedin a population.     

A 'large infrequent disturbance' in an East African savanna

There is growing interest in large infrequent disturbances (LIDs), but by definition they occur rarely and long‐term data are needed in order to study their effects and frequency. Palaeoecological records have the potential to provide information on the effects and frequency of LIDs. By comparing recent sedimentary records with known historical data, the effects of LIDs on pollen, charcoal and sedimentary sequences can be assessed. In this study, a LID in East Africa is described, and its representation in the palaeoecological record is explored. Historical records show that there was severe drought and famine in East Africa at the end of the 19th century. Fossil pollen and charcoal records from this period show evidence of a disturbance event that occurred at approximately this time. Statistical comparison of pollen and charcoal data from before, during and after the disturbance event identified it as a LID. The data also suggest that an erosion event occurred part way through the drought, indicating that an environmental threshold was exceeded.     

Nucleotide sequence of a type II DNA topoisomerase gene. Bacteriophage T4 gene 39.

T4 DNA topoisomerase is a type II enzyme and is thought to be required for normal T4 DNA replication T4 gene 39 codes for the largest of the three subunits of T4 DNA topoisomerase. I have determined the nucleotide sequence of a region of 2568 nucleotides of T4 DNA which includes gene 39. The location of the gene was established by the identification of the first fifteen amino acids in the large open reading frame in the DNA sequence as those found at the amino-terminus of the purified 39-protein. The coding region of gene 39 has 1560 bases, and it is followed by two in-frame stop codons. The gene is preceded by a typical Shine-Dalgarno sequence as well as possible promoter sequences for E. coli RNA polymerase. T4 39-protein consists of 520 amino acids, and it has a calculated molecular weight of 58,478. By comparing the amino acid sequences, T4 39-protein is found to share homology with the gyrB subunit of DNA gyrase. This suggests that these topoisomerase subunits may be equivalent functionally. Some of the characteristics of the 39-protein and its structural features predicted from the DNA sequence data are discussed.     

Terbium as a fluorescent probe for DNA and chromatin.

Terbium reacted with DNA and chromatin to form a complex in which terbium acted as a sensitive fluorescent probe. By measuring the narrow-line emission of Tb-3+ when DNA is selectively excited, the relative amount of Tb-3+ bound to the DNA can be calculated. Terbium was bound to DNA until one Tb-3+ was present for each phosphate group. After this point no more terbium was bound. TbCl3 was bound to chromatin in a linear manner until approximately 0.48 TbCl3 was added for each phosphate group in the chromatin-DNA solution. From these data it appears that 52% of the phosphate groups in chromatin were unavailable for binding. The binding of Tb-3+ to DNA can be reversed by prolonged dialysis against 0.5 M NaCl and chelating agents. The terbium ion is ideal in that it binds DNA tight enough so that completion of the reaction can be assumed but loose enough so that it can be removed by gentle means. Low concentrations of salt (up to 2 mM NaCl) enhance the quantum efficiency. Below pH 3 and above pH 7 the DNA-terbium complex will not form. Between pH 3 and pH 7 the quantum efficiency of the DNA terbium complex increases from either pH to a maximum at pH 5.5 to 5.6. Several biochemical uses for Tb-3+ ion are suggested.     

Neutral sucrose sedimentation of very large DNA from Bacillus subtilis. I. Effect of random double-strand breaks and centrifuge speed on sedimentation

A method is presented for preparing very large DNA from Bacillus subtilis protoplasts. When the DNA is characterized by sedimentation in neutral sucrose gradients, a fast-sedimenting component is found whose sedimentation coefficient varies with centrifuge speed. By use of [³H]thymine label for the DNA and a ¹⁴C-labeled amino acid, it is shown that less than 5% cellular material other than DNA is associated with this component. Irradiation of this DNA in solution with gamma rays forms a slower component, called the “main peak”, whose sedimentation coefficient also depends on centrifuge speed. More irradiation breaks down this main peak into even slower-sedimenting DNA; it is shown that for low doses, double-strand breaks are formed in both the B. subtilis DNA and in bacteriophage T2 DNA at the same rate linear in dose, 0.018 double-strand breaks per kilorad per mass equal to that of T2 DNA.The speed dependence of the DNA sedimenting at the main peak is compared with an approximate theory of the speed dependence of the sedimentation coefficient of linear DNA by B. H. Zimm (unpublished calculations). The comparison suggests that for sufficiently high centripedal acceleration, DNA molecules larger than a critical mass will sediment at much the same velocity. The theory, and data on the break-up of the DNA with gamma rays, are used to estimate that the DNA extracted is at least 13 times the mass of T2 DNA, and possibly larger.In the Appendix, data from the literature are put together with data taken during this work to make plausible the assumption that the usual theory for the sedimentation of DNA molecules, experimentally tested in salt solutions, may also be applied to sucrose solutions. If, in neutral sucrose gradients, the distance sedimented is proportional to a power α of the mass, the best value of α = 0.38.     

Science,truth, and forensic cultures: The exceptional legal status of DNA evidence

Many epistemological terms, such as investigation, inquiry, argument, evidence, and fact were established in law well before being associated with science. However, while legal proof remained qualified by standards of ‘moral certainty’, scientific proof attained a reputation for objectivity. Although most forms of legal evidence (including expert evidence) continue to be treated as fallible ‘opinions’ rather than objective ‘facts’, forensic DNA evidence increasingly is being granted an exceptional factual status. It did not always enjoy such status. Two decades ago, the scientific status of forensic DNA evidence was challenged in the scientific literature and in courts of law, but by the late 1990s it was being granted exceptional legal status. This paper reviews the ascendancy of DNA profiling, and argues that its widely-heralded objective status is bound up with systems of administrative accountability. The ‘administrative objectivity’ of DNA evidence rests upon observable and reportable bureaucratic rules, records, recording devices, protocols, and architectural arrangements. By highlighting administrative sources of objectivity, this paper suggests that DNA evidence remains bound within the context of ordinary organisational and practical routines, and is not a transcendent source of ‘truth’ in the criminal justice system.     

Complete elimination of mitosis and DNA synthesis in the lens of the hypophysectomized frog: effects on cell migration and fiber growth.

Three weeks after hypophysectomy mitosis and DNA synthesis are absent in the lens of the leopard frog (Rana iiens). By labeling germinative zone cells in their last DNA synthetic period we were able to follow them in the absence of further proliferation. By this means it has been demonstrated that migration of epithelial cells to the equator of the lens is stopped. Based on indirect measurements it seems that lens fiber formation also ceases. A correlation coefficient of 0.86 was computed between average labeling index and average distance migrated. In hypophysectomized animals the correlation coefficient between time and migration was 0.02 while in intact animals it is 0.87.     

Violations of the ceiling principle: exact conditions and statistical evidence.

The National Research Council recommended the use of the ceiling principle in forensic applications of DNA testing on the grounds that the ceiling principle was believed to be "conservative," giving estimates greater than or equal to the actual genotype frequencies in the appropriate reference population. We show here that the ceiling principle can fail to be conservative in a population with two subpopulations and two loci, each with two alleles at Hardy-Weinberg equilibrium, if there is some linkage disequilibrium between loci. We also show that the ceiling principle can fail in a population with two subpopulations and a single locus with two alleles if Hardy-Weinberg equilibrium does not hold. We give explicit analytical formulas to describe when the ceiling principle fails. By showing that the ceiling principle is not always mathematically reliable, this analysis gives users of the ceiling principle the responsibility of demonstrating that it is conservative for the particular data with which it is used. Our reanalysis of VNTR data bases of the FBI provides compelling evidence of two-locus associations within three major ethnic groups (Caucasian, black, and Hispanic) in the United States, even though the loci tested are located on different chromosomes. Before the ceiling principle is implemented, more research should be done to determine whether it may be violated in practice.     

Structure of the DNA-SspC complex: implications for DNA packaging, protection, and repair in bacterial spores

Bacterial spores have long been recognized as the sturdiest known life forms on earth, revealing extraordinary resistance to a broad range of environmental assaults. A family of highly conserved spore-specific DNA-binding proteins, termed alpha/beta-type small, acid-soluble spore proteins (SASP), plays a major role in mediating spore resistance. The mechanism by which these proteins exert their protective activity remains poorly understood, in part due to the lack of structural data on the DNA-SASP complex. By using cryoelectron microscopy, we have determined the structure of the helical complex formed between DNA and SspC, a characteristic member of the alpha/beta-type SASP family. The protein is found to fully coat the DNA, forming distinct protruding domains, and to modify DNA structure such that it adopts a 3.2-nm pitch. The protruding SspC motifs allow for interdigitation of adjacent DNA-SspC filaments into a tightly packed assembly of nucleoprotein helices. By effectively sequestering DNA molecules, this dense assembly of filaments is proposed to enhance and complement DNA protection obtained by DNA saturation with the alpha/beta-type SASP.     

Character analysis and the integration of molecular and morphological data in an understanding of sand dollar phylogeny.

Reconciling discordant morphological and molecular phylogenies remains a problem in modern systematics. By examining conflicting DNA-hybridization and morphological phylogenies of sand dollars, I show that morphological criteria may be used to help evaluate the reliability of molecular phylogenies where they differ from morphological trees. All available criteria for assessing the reliability of DNA-hybridization phylogenies suggest that the sand dollar DNA-hybridization phylogeny is robust. Standard homology-recognition criteria are used to assess the a priori reliabilities of the morphological attributes associated with the node drawn into question by the DNA data, and it is shown that these attributes are among the least phylogenetically informative of all the morphological characters. Moreover, the questioned node has the smallest number of supporting characters, and most of these characters are associated with the food grooves, which suggests that they may be functionally correlated. Thus, on the basis of the analysis of the morphological data and given the robustness of the DNA tree, the DNA phylogeny is preferred. Further, paleobiogeographic data support the DNA tree rather than the morphological tree, and a plausible heterochronic mechanism has been proposed that may account for the homoplasious morphological evolution that must have occurred if the DNA tree is correct.