The DNA binding of insect Fork head factors is strongly influenced by the negative cooperation of neighbouring bases

The Drosophila melanogaster Fork head and Bombyx mori SGF1/Fork head proteins are key regulators of tissue specific gene expression in the modified larval labial glands. Here we use the competitive electrophoretic mobility shift assay to create a detailed Fork head binding matrix and we investigate some unusual features of the Fork head interaction with DNA. We found that the Fork head-DNA interaction is context dependent--the binding specificity of the protein is partly determined by specific combinations of neighbouring bases. Although the total number of the sub-optimal dinucleotide steps is not high, the negative cooperation of neighbouring bases significantly contributes to the overall binding site specificity. Our results allow efficient recognition of insect Fork head binding sites and we show that the putative Fork head cognate elements preferentially accumulate in the near upstream region of genes abundantly expressed in the labial gland.     

The Drosophila fork head factor directly controls larval salivary gland-specific expression of the glue protein gene Sgs3.

The Drosophila Fork head protein participates in salivary gland formation, since salivary glands are missing in fork head embryos. Here we show that the fork head encoded protein binds to an upstream regulatory region of the larval salivary gland glue protein gene Sgs3. Mobility shift assay in the presence of an anti-Fork head antibody demonstrated that the Fork head factor interacts with the TGTTTGC box shown to be involved in tissue-specific Sgs3 expression. Experiments employing a set of oligonucleotide competitors revealed that Fork head binding was prevented by the same single base substitutions that were previously shown to interfere with the TGTTTGC element function in vivo. Furthermore, the anti-Fork head antibody bound to >60 sites of polytene chromosomes, including the puffs of all Sgs genes and Fork head protein was detected in the nuclei of salivary glands of larvae of all examined stages. These data provide experimental evidence for the hypothesis that the protein encoded by the fork head gene is required initially for salivary gland formation and is utilized subsequently in the control of larval genes specifically expressed in this organ.     

DISTAN -- A program which detects significant distances between short oligonucleotides

We present an algorithm to detect distances between oligonucleotidesin large collections of nucleic acids sequences. The ratiosof actual frequencies of occurrence of short oligonucleotidesat a given distance to the corresponding expected frequencieswere analyzed in four categories of DNA sequences (eukaryoticexons, bacterial genes, introns and non-Alu repeated DNAs).Three base periodic occurrences (independent of the readingframe) of all combinations of mononucleotides and repeats ofall dinucleotides was characteristic for protein coding regions.This was also the case with the majority of trinucleotides (includingtranslational stop signals) in these regions. Mirror-symmetrictrinucleotides (except GCG and CGC) displayed a strong tendencyto be two base periodically repeated in introns. Some two andthree base periodic motifs were also observed in repeated DNAs.The possible biological implications of outstanding three baseperiodicities in bacterial genes and eukaryotic exons are discussed. Received on March 2, 1987; accepted on May 5, 1987     

N-acetyltransferase 1 and 2 gene sequence variants and risk of head and neck cancer

Polymorphisms that alter the function of genes involved in the activation or detoxification of carcinogenic compounds can influence an individuals risk of developing cancer. Polymorphic changes modulating the acetylation capacity of the N-acetyltransferase (NAT) genes have been implicated in the risk of developing cancer. In this study the role of genetically determined individual NAT1 and NAT2 genotypes, haplotypes and haplotype combinations in the predisposition to head and neck cancer was investigated. Polymorphic regions of the NAT1 and NAT2 genes were analyzed in patients with head and neck cancer and healthy individuals by polymerase chain reaction-restriction fragment length polymorphism. Distribution of the genotypes, allele frequencies, diplotypes and haplotypes and correlation with clinical characteristics were evaluated. No association was observed between the NAT1*3, NAT1*10, NAT1*11, NAT2*5 and NAT2*6 genotypes and risk of head and neck cancer. The NAT2*7 slow genotype was associated with reduced risk of disease. A significant association was observed between the fast acetylator NAT2*4/NAT1*10 diplotype and risk of head and neck cancer. Combined haplotypes harboring the T1088A and C1095A variants characterizing the NAT1*10 allele were associated with increased risk. Our results suggest that NAT1 and NAT2 gene combinations may influence the risk of developing head and neck cancer.     

Melting profile and temperature dependent binding constant of an anticancer drug daunomycin-DNA complex

We calculate thermal fluctuational base pair opening probability and the drug binding constant of a daunomycin-bound Poly d(CGTA) · Poly d(TACG) at temperatures from room temperature to its melting temperature. For comparison we also carry out a calculation on a drug-free DNA with the same sequence. Our calculations are carried out by means of a statistical approach using microscopic structures and established force fields and with cooperative effects incorporated into the algorithm. Both hydrogen bond disruption probabilities and drug unstacking probability are determined self-consistently. These probabilities are then used to determine temperature dependent base pair opening probabilities and the drug binding constant. The calculated base pair opening probabilities and drug binding constant are found to be in fair agreement with experiments carried out at room temperature. Our calculation shows cooperative base pair disruption and drug dissociation at certain critical temperatures close to the observed melting temperatures for similar helices. We find that the temperature dependence of the drug binding constant fits well to the van't Hoff relation, in agreement with observations. Our calculation indicates the occurrence of a premelting transition in the drug-bound DNA helix. Some comments are made about this premelting transition.     

Nearest-neighbor tree species combinations in tropical forest: the role of chance, and some consequences of high diversity

In three permanent inventory plots comprising 12.4 ha of undisturbed forest at La Selva, Costa Rica, all stems ≥10 cm dbh were mapped and identified to species. There were 1628, 1478 and 1954 trees in the plots, representing 168, 166 and 171 species respectively. We determined the species of each nearest-neighbor pair of trees, and asked whether the occurrence of species pairs conforms to a simple random mixing model. If trees are randomly mixed in terms of species, the expected frequency of any nearest neighbor species combination is a function of the relative abundance of the two species. Departures from random mixing could arise from species interactions, differential responses to habitat, or both. The number of possible ij species combinations increases approximately as the square of the number of species. For the 168 species in plot 1, for example, there are 14 196 possible combinations. We compared the expected frequency of each species combination in the three plots (42 736 combinations in all) with observed frequencies. Over 98% of the combinations had observed frequencies of zero and expected frequencies close to zero. A consequence of high diversity is low density of most individual species, and exceedingly low frequencies of the vast majority of species combinations. For each of the 805 combinations with observed frequencies >0, we used simulation to generate a distribution of expected frequencies. We used a t-test to compare the observed frequency with the mean of the simulated distribution for each combination. Only 40 combinations (0.09% of the possible species combinations in the plots) departed from expected frequencies; 39 combinations were more common, and one less common than expected. The overwhelming majority of nearest neighbor species combinations occur at frequencies predictable from their individual abundances.     

Evolution of Microsatellites in the Yeast Saccharomyces cerevisiae: Role of Length and Number of Repeated Units

The observed and expected frequencies of occurrence of microsatellites in the yeast Saccharomyces cerevisiae were investigated. In all cases, the observed frequencies exceeded the expected ones. In contrast to predictions by Messier et al. (1996), there is no critical number of repeats beyond which the observed frequencies of microsatellites significantly exceed the frequencies expected in a random DNA sequence of the same size. Rather, the degree of deviation from expectation was found to be dependent on the length of the microsatellite. That is, a fourfold concatemeric repeat of 3 bp was found to deviate from expectation as much as threefold concatemeric repeat of 4 bp, unlike the deviation of a fourfold concatemeric repeat of 4 bp. These findings suggest that microsatellites evolve through strand-slippage events, rather than recombination events. This, in turn, suggests that the chances of erroneous hybridizations leading to strand-slippage are length dependent. Received: 1 June 1998 / Accepted: 16 September 1998     

Longitudinal movement of fish in response to a single-day flow pulse

We studied whether fish were displaced longitudinally downstream by a 1-day whitewater kayaking pulse flow release from Camino Dam on Silver Creek, a tributary of the South Fork American River, California. On 15 September 2004 flows were increased from a base flow of 0.48 m³·s^-1 to a peak of 18.48 m³·s^-1 by midday, and decreased back to base levels. Rainbow trout (Oncorhynchus mykiss) and brown trout (Salmo trutta) were observed in snorkel surveys before and after the pulse. Counts of young-of-the-year and juvenile trout were 26% and 9% lower after the pulse, respectively. Counts of adult trout were 12% higher. Six adult trout were radio-tagged and were observed in the reach before, during, and after the pulsed flow. Our results suggest that most trout were able to remain in the study reach during the pulse, but that smaller fish may be more likely to be displaced downstream.     

The effects of Cu2+ and Pb2+ on the solution structure of calf thymus DNA: DNA condensation and denaturation studied by fourier transform IR difference spectroscopy

The interaction of calf thymus DNA with Cu²⁺and Pb²⁺ was studied in aqueous solution at pH 6.5 with metal/DNA (P) (P = phosphate) molar ratios (r) 1/80, 1/40, 1/20, 1/10, 1/4, 1/2, and 1, using Fourier Transform ir (FTIR) spectroscopy. Correlations between the ir spectral changes, metal ion binding mode, DNA condensation, and denaturation, as well as conformational features, were established. Spectroscopic evidence has shown that at low metal/DNA (P) molar rations 1/80 and 1/40, copper and lead ions bind mainly to the PO of the backbone, resulting in increased base-stacking interaction and duplex stability. The major copper ion base binding via G-C base pairs begins at r > 1/40, while the lead ion base binding occurs at r > 1/20 with the A-T base pairs. The denaturation of DNA begins at r = 1/10 and continues up to r = 1/2 in the presence of copper ions, whereas a partial destabilization of the helical structure was observed for the lead ion at high metal ion concentration (r = 1/2). Metal-DNA binding also results in DNA condensation. No major departure from the B-family structure was observed, upon DNA interaction with these metal ions. © 1993 John Wiley & Sons, Inc.     

Interaction of spermine and DNA

The effect of spermine upon the denaturation temperature (T_m) of DNA's of various base compositions has been found to depend upon both the base composition of the DNA and the pH of the solution. Measurement of the hydrogen ion titration curve of spermine as a function of temperature reveals that the net charge of the spermine molecule is undergoing a rapid change with temperature in the range of temperatures at which DNA denatures. Since the value of T_m depends upon base composition, the correlation of the effect of spermine upon T_m with the base composition of the DNA used may be explainable in terms of the changing degree of ionization of spermine. The binding of spermine to native DNA has also been studied by dialysis equilibrium. There is no significant variation either in the number of strongly binding sites or strength of binding with base composition. It is concluded that there is no evidence of correlation between the binding of spermine and the base composition of DNA.     

87 The melting of DNA in the presence of meso-tetra-pyridyl porphyrins with different peripheral substituents

The influence of water-soluble cationic 3N- and 4N-pyridyl porphyrins with different peripheral substituents (oxyethyl, buthyl, allyl, and metallyl) on melting parameters of DNA has been studied. Results indicate that the presence of porphyrin changes the shape and parameters of DNA melting curve. The increase of porphyrins concentration results in the increase of the melting temperature (Tm) and the melting interval (ΔT) of DNA. At the porphyrin-DNA concentration ratio r?=?0.01, changes in the melting temperature have not been observed. The melting intervals almost do not change upon adding of the 4N-porphyrins, while the decrease of ΔT, in the presence of 3N-porphyrins, is observed. Because the intercalation binding mechanism occurs in GC-rich regions of DNA, we assume that 3N-porphyrins, intercalated in GC-rich regions, reduce the thermal stability of these sites, bringing them closer to the thermal stability of the AT-sites, which is the reason for the decrease in the melting interval. While at the relative concentration r?=?0.01 for 4-N porphyrins, already the external binding mechanism “turns on” and the destabilizing effect of porphyrins on GC-pairs compensates stabilizing effect on AT-pairs, as a result of which change in the melting of DNA upon complexation with these porphyrins is not observed. The decrease of the hypochromic effect also indicates the intercalation of investigated porphyrins in the DNA structure, which weakens the staking interaction of base pairs of DNA. The increase of the hypochromic effect of DNA upon binding with porphyrin depends on the type of peripheral substituents of the porphyrin. The results show that porphyrins with butyl and allyl substituents weaken staking interaction of base pairs less than porphyrins with other substituents. The largest change was observed for metallyl porphyrins. It can be the result of bulky peripheral substituents, which make significant local changes in DNA structure.     

A new design without control population for identification of gastric cancer-related allele combinations based on interaction of genes

Objective

The purpose of this study was to develop a novel approach without control population to examine the relationship between the presence of specific allele combinations at different loci with the onset of gastric cancer.

Methods

DNA samples were collected from patients with gastric cancer. Alleles from short tandem repeat loci were determined using the STR Profiler Plus PCR amplification kit (15 STR loci). The observed and expected frequencies of specific allele combinations were calculated; statistically significant allele combinations were identified by comparing the observed frequency with the expected frequency. The age at disease onset of patients carrying a specific allele combination was further analysed; allele combinations related to the gastric cancer were effectively identified from the large number of possible allele combinations by cross-validation of the 2 sets of analytical results.

Results

A total of 2209 pairwise combinations were obtained by computer counting, of which 11 pairs of genes showed significant differences between the observed and expected frequencies (p < 0.05). The p value for the cross-validation was also less than 0.05 for 2 pair of alleles (D8S1179-16 and D5S818-13; D2S1338-23 and D6S1043-11).

Conclusion

Gastric cancer onset may be associated with these allele combinations. The new methodology without control group will enable additional discoveries pertaining to the relationship between specific allele combinations at different loci and the onset of complex diseases.     

Specific protection of DNA by distamycin A, netropsin and bis-netropsins against the action of DNAse I

Interaction of netropsin, distamycin A and a number of bis-netropsins with DNA fragments of definite nucleotide sequence was studied by footprinting technique. The nuclease protection experiments were made at fixed DNA concentration and varying ligand concentrations. The affinity of ligand for a DNA site was estimated from measurements of ligand concentration that causes 50% protection of the DNA site. Distribution pattern of the protected and unprotected regions along the DNA fragment was compared with the theoretically expected arrangement of the ligand along the same DNA. The comparison led us to the following conclusions: 1. Footprinting experiments show that at high levels of binding the arrangement of netropsin molecules along the DNA corresponds closely to the distribution pattern expected from theoretical calculations based on the known geometry of netropsin--DNA complex. However, the observed differences in the affinity of netropsin for various DNA sequences is markedly greater than that expected from theoretical calculations. 2. Netropsin exhibits a greater selectivity of binding than that expected for a ligand with three specific reaction centers associated with the antibiotic amide groups. It binds preferentially to DNA regions containing four or more successive AT pairs. Among 13 putative binding sites for netropsin with four or more successive AT pairs there are 11 strong binding sites and two weaker sites which are occupied at 2 D/P less than or equal to 1/9 and 2 D/P = 1/4, respectively. 3. The extent of specificity manifested by distamycin A is comparable to that shown by netropsin although the molecule of distamycin A contains four rather than three amide groups. At high levels of binding distamycin A occupies the same binding sites on DNA as netropsin does. 4. The binding specificity of bis-netropsins is greater than that of netropsin. Bis-netropsins can bind to DNA in such a way that the two netropsin-like fragments are implicated in specific interaction with DNA base pairs. However, the apparent affinity of bis-netropsins estimated from footprinting experiments is comparable with that of netropsin for the same DNA region. 5. At high levels of binding bis-netropsins and distamycin A (but not netropsin) can occupy any potential site on DNA irrespectively of the DNA sequence. 6. Complex formation with netropsin increases sensitivity to DNase I at certain DNA sites along with the protection effect observed at neighboring sites.     

Spatiotemporal assessment of Sickle Darter (Percina williamsi Page and Near, 2007) distribution in the upper Tennessee River Basin

The Sickle Darter Percina williamsi (Page and Near, 2007) is a species of fish endemic to the upper Tennessee River basin in eastern Tennessee, southwestern Virginia, and western North Carolina. Because of its narrow range and presumed decline in occupied sites over the last half century, it is being proposed for federal listing under the Endangered Species Act. We analyzed the current distribution of the Sickle Darter and temporal trends in its distribution in relation to temporal trends in environmental and habitat covariates for each of the historically occupied sub-basins (upper Clinch, Emory, upper French Broad, Little, Little Pigeon, Middle Fork Holston, North Fork Holston, Powell, South Fork Holston, and Watauga) with multiple linear regression modelling. A total of 154 Sickle Darters were observed at 15 sites throughout the upper Tennessee River Basin. Sickle Darters were observed in the Little River, Emory River, and Middle Fork Holston River sub-basins. A total of 133 unique historical occurrences were used for the spatiotemporal analyses. Sickle Darters have declined in 8 out of 10 historically occupied sub-basins. Our best model for the whole distribution scale (Mallow's Cp = −0.87; Adjusted R² = .92) suggests that habitat fragmentation due to damming has had adverse effects on Sickle Darter populations across its distribution. Models were very similar for the sub-basin specific models as well. The results from this study highlight the drivers of decline in Sickle Darter distribution and outline the future research needs for this species that should be used to inform future conservation decisions regarding this species.     

The Codon-Degeneracy Model of Molecular Evolution

Mitochondrial genetic codons can be categorized by four patterns of nucleotide-site degeneracy based on varying combinations of twofold- or nondegenerate sites at first codon positions and twofold- or fourfold-degenerate sites at third codon positions. Herein, a model of molecular evolution is introduced that uses these patterns to calculate expected substitution frequencies for each codon position and substitution type relative to overall number of synonymous or nonsynonymous substitutions. Regions of the pocket gopher cytochrome oxidase subunit I (COI) and cytochrome b (cyt-b) genes are analyzed using this model. Chi-square distributions are used to produce relative goodness-of-fit (GF) scores for measuring the difference between substitution frequencies predicted by the codon-degeneracy model (CDM), and frequencies inferred using a well-supported phylogenetic tree of closely related species. The GF scores for expected and observed synonymous (GF_syn= 0.429, p= 0.807) and nonsynonymous (GF_ns= 2.309, p= 0.679) substitution frequencies resulted in a failure to reject the CDM as a null hypothesis for the molecular evolution of COI and cyt-b in pocket gophers. Alternative tree topologies and calculations of transition bias for these data result in higher GF scores. Received: 25 March 1999 / Accepted: 17 September 1999     

Analysis of cooperativity and ion effects in the interaction of quinacrine with DNA

The interaction of quinacrine with calf thymus DNA was monitored at several different ionic strengths using spectrophotometric and equilibrium dialysis techniques. The binding results can be explained, assuming each base pair is a potential binding site, using a model containing two negative cooperative effects: (1) ligand exclusion at binding sites adjacent to a filled binding site and (2) ligand–ligand negative cooperativity at adjacent filled binding sites. The logarithm of the observed equilibrium constant (K_obs) determined by this model varies linearily with log[Na⁺], as predicted by the ion condensation theory for polyelectrolytes. When the log K_obs plot is correlated for sodium release by DNA in the intercalation conformational change, the predicted number of ion pairs between the ligand and DNA is approximately two, as expected for the quinacrine dication. Even though K_obs depends strongly on ionic strength, the ligand negative cooperativity parameter ω was found to be indpendent of ionic strength within experimental error. This finding is also in agreement with the ion condensation theory, which predicts a relatively constant amount of condensed counterion on the DNA double helix over this ionic strength range. Drugs would, therefore, experience a relatively constant ionic environment when complexed to DNA even though the ionic conditions of the solvent could change considerably.     

Symbiont infection affects whitefly dynamics in the field

Inherited intracellular insect endosymbionts may manipulate host reproduction or provide fitness benefits to their hosts in ways that result in their rapid spread throughout a host population. Fitness benefits in particular can result in the increased pest potential of agriculturally important insects. While benefits due to endosymbiont infection have been well studied in laboratory assays, very little is known about how these benefits translate to insect performance in the field. Laboratory experiments have shown that the maternally inherited bacterial endosymbiont, Rickettsia, increases the fitness of its whitefly host, Bemisia tabaci, through improved fecundity, faster development times and female-biased sex ratios. We conducted field population cage studies to determine whether the benefits conferred by Rickettsia to whiteflies in the laboratory were evident on one of its major hosts, cotton, under field conditions in Arizona, USA. In cages with either Rickettsia-infected or uninfected whiteflies, we observed up to ten-fold higher whitefly egg and nymph densities when whiteflies were Rickettsia-infected compared with uninfected whiteflies throughout the season. We also observed a steep initial increase in Rickettsia frequency in population cages started with either 25% or 50% Rickettsia-infected whiteflies, with the 50% cages approaching fixation within three generations. Using growth rates obtained in the density cages, we calculated and compared an expected trajectory of the frequencies of Rickettsia infection with the observed frequencies. Results showed similar observed and expected frequencies of Rickettsia in the first two generations, followed by a significantly lower than expected frequency in three of four treatment/sample combinations at the end of the season. Taken together, these results confirm the patterns of fecundity and population growth observed in laboratory assays, under field conditions, as well as provides preliminary empirical support for a Rickettsia equilibrium frequency of less than 100%.     

Does DNA absorb microwave energy?

The microwave absorption of chicken erythrocytes and E. coli DNA aqueous solutions was studied in the 9–12-GHz frequency range by the method of variable thickness. At the same frequencies, fragments of sonicated erythrocyte DNA (average size about 500 base pairs) and of E. coli DNA treated with DNase (most of which were 800–2800 base pairs) were investigated. In neither case was any effect of enhanced microwave absorption by DNA observed. It was shown that an excess absorption of DNA solution falls within the 1% experimental error range, provided the conductivity contribution of 1% MgCl₂, required for DNase action, to the microwave absorption is taken into account.