Steady-state and rapid kinetic analysis of topoisomerase II trapped as the closed-clamp intermediate by ICRF-193

DNA topoisomerase II uses a complex, sequential mechanism of ATP hydrolysis to catalyze the transport of one DNA duplex through a transient break in another. ICRF-193 is a catalytic inhibitor of topoisomerase II that is known to trap a closed-clamp intermediate form of the enzyme. Using steady-state and rapid kinetic ATPase and DNA transport assays, we have analyzed how trapping this intermediate by the drug perturbs the topoisomerase II mechanism. The drug has no effect on the rate of the first turnover of decatenation but potently inhibits subsequent turnovers with an IC(50) of 6.5 +/- 1 microM for the Saccharomyces cerevisiae enzyme. This drug inhibits the ATPase activity of topoisomerase II by an unusual, mixed-type mechanism; the drug is not a competitive inhibitor of ATP, and even at saturating concentrations of drug, the enzyme continues to hydrolyze ATP, albeit at a reduced rate. Topoisomerase II that was specifically isolated in the drug-bound, closed-clamp form continues to hydrolyze ATP, indicating that the enzyme clamp does not need to re-open to bind and hydrolyze ATP. When rapid-quench ATPase assays were initiated by the addition of ATP, the drug had no effect on the sequential hydrolysis of either the first or second ATP. By contrast, when the drug was prebound, the enzyme hydrolyzed one labeled ATP at the uninhibited rate but did not hydrolyze a second ATP. These results are interpreted in terms of the catalytic mechanism for topoisomerase II and suggest that ICRF-193 interacts with the enzyme bound to one ADP.     

The rug3 locus of pea encodes plastidial phosphoglucomutase

Two cDNA clones were isolated from pea (Pisum sativum L.) and their deduced amino acid sequences shown to have significant homology to phosphoglucomutases from eukaryotic and prokaryotic sources. The longer cDNA contained a putative transit-peptide-encoding sequence, supporting the hypothesis that the isolated clones represent the cytosolic and plastidial isoforms of phosphoglucomutase in pea. Plastid protein import assays confirmed that the putative plastidial isoform was targeted to the plastid stroma where it was proteolytically processed. Expression, co-segregation, linkage, and molecular analyses have confirmed that the rug3 locus of pea encodes plastidial phosphoglucomutase. Mutations at this locus result in a near-starchless phenotype of the plant.     

High levels of sequence polymorphism and linkage disequilibrium at the telomere of 12q: implications for telomere biology and human evolution

The human Xp/Yp telomere-junction region exhibits high levels of sequence polymorphism and linkage disequilibrium. To determine whether this is a general feature of human telomeres, we have undertaken sequence analysis at the 12q telomere and have extended the analysis at Xp/Yp. A total of 22 single-nucleotide polymorphisms (SNPs) and one 30-bp duplication were detected in the 1,870 bp adjacent to the 12q telomere. Twenty polymorphic positions were in almost complete linkage disequilibrium, creating three common diverged haplotypes accounting for 80% of 12q telomeres in the white population. A further 6% of 12q telomeres contained a 1,439-bp deletion in the DNA flanking the telomere. The remaining 13% of 12q telomeres did not amplify with the primers used (nulls). The distribution of telomere (TTAGGG) and variant repeats within 12q telomeres was hypervariable, but alleles with similar distribution patterns were associated with the same haplotype in the telomere-adjacent DNA. These data suggest that 12q telomeres, like Xp/Yp telomeres, exhibit low levels of homologous recombination and evolve along haploid lineages. In contrast, high levels of homologous recombination occur in the adjacent proterminal regions of human chromosomes. This suggests that there is a localized telomere-mediated suppression of recombination. In addition, the genetic characteristics of these regions may provide a source of deep lineages for the study of early human evolution, unaffected by both natural selection and recombination. To explain the presence of a few diverged haplotypes adjacent to the Xp/Yp and 12q telomeres, we propose a model that involves the hybridization of two archaic hominoid lineages ultimately giving rise to modern Homo sapiens.     

Structural effects of DNA sequence on T.A recognition by hydroxypyrrole/pyrrole pairs in the minor groove

Synthetic polyamides composed of three types of aromatic amino acids, N-methylimidazole (Im), N-methylpyrrole (Py) and N-methyl-3-hydroxypyrrole (Hp) bind specific DNA sequences as antiparallel dimers in the minor groove. The side-by-side pairings of aromatic rings in the dimer afford a general recognition code that allows all four base-pairs to be distinguished. To examine the structural consequences of changing the DNA sequence context on T.A recognition by Hp/Py pairs in the minor groove, crystal structures of polyamide dimers (ImPyHpPy)(2) and the pyrrole counterpart (ImPyPyPy)(2) bound to the six base-pair target site 5'-AGATCT-3' in a ten base-pair oligonucleotide have been determined to a resolution of 2.27 and 2.15 A, respectively. The structures demonstrate that the principles of Hp/Py recognition of T.A are consistent between different sequence contexts. However, a general structural explanation for the non-additive reduction in binding affinity due to introduction of the hydroxyl group is less clear. Comparison with other polyamide-DNA cocrystal structures reveals structural themes and differences that may relate to sequence preference.     

Accounting for Missing Data in Noncancer Risk Assessment

Noncancer risk assessments are generally forced to rely on animal bioassay data to estimate a Tolerable Daily Intake or Reference Dose, as a proxy for the threshold of human response. In cases where animal bioassays are missing from a complete data base, the critical NOAEL (no-observed-adverse-effect level) needs to be adjusted to account for the impact of the missing bioassay(s). This paper presents two approaches for making such adjustments. One is based on regression analysis and seeks to provide a point estimate of the adjustment needed. The other relies on non-parametric analysis and is intended to provide a distributional estimate of the needed adjustment. The adjustment needed is dependent on the definition of a complete data base, the number of bioassays missing, the specific bioassays which are missing, and the method used for interspecies scaling. The results from either approach can be used in conjunction with current practices for computing the TDI or RfD, or as an element of distributional approaches for estimating the human population threshold.     

Identification of a QTL decreasing yield in barley linked to Mlo powdery mildew resistance

A molecular marker map, including Mlo mildew resistance, of the spring barley cross Derkado (Mlo-resistant) × B83-12/21/5 (Mlo-susceptible) was scanned for yield QTLs to determine whether the association of Mlo resistance with reduced yield was due to linkage or pleiotropy. Over the mapped portion of the genome of the cross, the QTL with the greatest effect upon yield was located within a 22 cM region between mlo and the simple sequence repeat HVM67 on chromosome 4(4H). The association of Mlo resistance with lower yield was therefore due to a repulsion linkage. Analysis of yield component characters revealed QTL alleles for reduced grain number and earlier heading date in the same region, also associated with Mlo resistance. Genotyping of a range of cultivars and sources of Mlo resistance with the HVM67 simple sequence repeat showed that the Derkado HVM67 allele was rare as it was found only in one other cultivar and four land-races or sources of disease resistance. Grannenlose Zweizeilige, the source, and Salome, the carrier of Mlo resistance in Derkado, have the same HVM67 genotype, although Salome was a mixture of two genotypes. The entire mlo-HVM67 chromosomal segment from Grannenlose Zweizeilige is therefore thought to have been transmitted to Derkado, possibly through joint selection for Mlo resistance and early heading. L92, synonym EP79, was another source of Mlo resistance with the same HVM67 allele as Derkado but recombination must have occurred during the breeding of Atem as it possesses a different HVM67 allele which is present in all the other Mlo sources and cultivars surveyed. Abbreviations: GN, grains per main stem ear; HD, heading date; MSTGW, thousand grain weight derived from GN and MSY; MSY, yield of grain on the main stem; PY, yield of grain from the whole plot; sCIM, simplified compound interval mapping; SIM, simple interval mapping; SPY, single plant yield; S-SAP, sequence-specific amplification polymorphism; TGW, thousand grain weight derived from bulk of plot seed; TN, number of fertile stems per plant.     

A new heterospecific foraging association between the puddingwife wrasse,Halichoeres radiatus,and the bar jack,Caranx Tuber: evaluation of the foraging consequences

Synopsis The bar jack,Caranx Tuber, was commonly observed to follow individual puddingwife wrasses,Halichoeres radiatus, that were foraging on the substrate. Individuals of both species actively pursued the other to maintain these heterospecific foraging teams, were sometimes attracted to feeding acts initiated by team partners, and the foraging rates of teamed jacks and wrasses were positively correlated. Pilfering of food items was rare, suggesting little, if any, competition cost of this foraging association. The ratio of bites to search in teamed jacks was over three times that when solitary, and jacks were sometimes aggressive to conspecifics attempting to join their team, suggesting that the association is beneficial to the jacks. Both bite and search rates were higher in puddingwifes when teamed with a jack, indicating that the association also benefits the wrasses. Benefits to puddingwifes may be derived directly from attendants because wrasses were sometimes attracted to jack foraging acts. However, increased foraging in wrasses may also be a consequence of heightened motivation to feed owing to heterospecific social facilitation.     

Effects of synthetic and naturally occurring flavonoids on metabolic activation of benzo[a]pyrene in hamster embryo cell cultures.

Biochanin A, an isoflavone, has previously been shown to inhibit the metabolic activation of the carcinogen benzo[a]pyrene (B[a]P) to metabolites that bind to DNA in hamster embryo cells and are mutagenic in Chinese hamster V79 cells. To determine the structural features required for this activity and to attempt to find more effective inhibitors, a series of synthetic and naturally occurring flavonids were tested for their ability to modulate B[a]P metabolism in hamster embryo cell cultures. The observed structure-activity relationships indicate that the structural features of flavonoids important for effective inhibition of B[a]P metabolism in hamster embryo cells are the presence of two hydroxyl, two methoxyl, or methyl and hydroxyl substituents at the 5- and 7-positions and a 2,3-double bond. Flavones are slightly better inhibitors of B[a]P metabolism than the corresponding isoflavones. A substituent at the 4'-position is not essential for inhibition of B bdP metabolism. The presence of a hydroxyl group at position 3 slightly enhances activity. Apigenin, acacetin and kaempferide are effective inhibitors of B[a]P-induced mutagenesis in a hamster embryo cell-mediated V79 cell mutation assay. However, apigenin is cytotoxic at the inhibitory dose, whereas acacetin and kaempferide are not. These results suggest that acacetin and kaempferide are promising candidates for in vivo testing as potential chemopreventive agents.