DNA polymerase V allows bypass of toxic guanine oxidation products in vivo

Reactive oxygen and nitrogen radicals produced during metabolic processes, such as respiration and inflammation, combine with DNA to form many lesions primarily at guanine sites. Understanding the roles of the polymerases responsible for the processing of these products to mutations could illuminate molecular mechanisms that correlate oxidative stress with cancer. Using M13 viral genomes engineered to contain single DNA lesions and Escherichia coli strains with specific polymerase (pol) knockouts, we show that pol V is required for efficient bypass of structurally diverse, highly mutagenic guanine oxidation products in vivo. We also find that pol IV participates in the bypass of two spiroiminodihydantoin lesions. Furthermore, we report that one lesion, 5-guanidino-4-nitroimidazole, is a substrate for multiple SOS polymerases, whereby pol II is necessary for error-free replication and pol V for error-prone replication past this lesion. The results spotlight a major role for pol V and minor roles for pol II and pol IV in the mechanism of guanine oxidation mutagenesis.     

Genetic diversity and demographic history of introduced sika deer on the Delmarva Peninsula

The introduction of non‐native species can have long‐term effects on native plant and animal communities. Introduced populations are occasionally not well understood and offer opportunities to evaluate changes in genetic structure through time and major population changes such as bottleneck and or founder events. Invasive species can often evolve rapidly in new and novel environments, which could be essential to their long‐term success. Sika deer are native to East Asia, and their introduction and establishment to the Delmarva Peninsula, USA, is poorly documented, but probably involved ≥1 founder and/or bottleneck events. We quantified neutral genetic diversity in the introduced population and compared genetic differentiation and diversity to the presumed source population from Yakushima Island, Japan, and a captive population of sika deer in Harrington, Delaware, USA. Based on the data from 10 microsatellite DNA loci, we observed reduced genetic variation attributable to founder events, support for historic hybridization events, and evidence that the population did originate from Yakushima Island stocks. Estimates of population structure through Bayesian clustering and demographic history derived from approximate Bayesian computation (ABC), were consistent with the hypothesized founder history of the introduced population in both timing and effective population size (approximately five effective breeding individuals, an estimated 36 generations ago). Our ABC results further supported a single introduction into the wild happening before sika deer spread throughout the Delmarva. We conclude that free‐ranging sika deer on Delmarva are descended from ca. five individuals introduced about 100 years ago from captive stocks of deer maintained in the United Kingdom. Free‐ranging sika deer on Delmarva have lost neutral diversity due to founder and bottleneck events, yet populations have expanded in recent decades and show no evidence of abnormalities associated with inbreeding. We suggest management practices including increasing harvest areas and specifically managing sika deer outside of Maryland.     

Selection for increased percentage phaseolin in common bean

Summary Two selection methods were compared to determine which was more efficient for increasing percentage phaseolin in the common bean (Phaseolus vulgaris L.). A base population consisting of families segregating for six seed protein alleles (Phas ^S , Phas ^C , Phas ^T , phas ^-, lec^-, and Arcl ⁺), all of which have measurable effects on percentage phaseolin, was subjected to either three cycles of S₁ family recurrent selection for increased percentage phaseolin (PPS), or one cycle of selection for combinations of the protein alleles (PAS) known to have positive effects on phaseolin accumulation. One cycle of PAS resulted in an increase in percentage phaseolin that was equivalent to three cycles of PPS. Selection under both methods produced increases in several correlated traits including percentage total protein, phaseolin as a percent of total protein, mg protein/seed, and mg phaseolin/seed. The amount of nonphaseolin protein per seed decreased, while seed yield was unaffected by either selection procedure. By selecting for favorable seed protein alleles identified by electrophoresis, it was possible to rapidly increase percentage phaseolin without the need for field evaluation.     

Alternative diaspirins for modification of hemoglobin and sickle hemoglobin

Studies of modification of hemoglobin and of sickle hemoglobin by alternative aspirins have been extended to a series of new bis esters with a variety of substituted bridging diacids and to a group of mono esters with polar acyl groups. Rates of hydrolysis of these alternative aspirins have also been examined, and they reveal that a careful balance between stability and reactivity is essential for optimal activity. Four-carbon bridging groups have been found to be particularly effective, two of these raising the minimum gelling concentration of sickle hemoglobin by as much as 100%.     

Cis-acting sequences sufficient for correct tissue and temporal specificity of larval serum protein 1 genes of Drosophila

We have constructed hybrid genes in which the coding region of the bacterial gene chloramphenicol acetyl transferase (CAT) has been linked to varying lengths of upstream sequences of Drosophila genes for larval serum sequence 1 (LSP1). These have been inserted into a P-element transformation vector and subsequently transferred into the germ-line of recipient flies. Transformants carrying the CAT gene linked to 1650 bp, 570 bp or 377 bp of upstream LSP1α sequences, or 745 bp or 471 bp of upstream β sequences express CAT with the same developmental and tissue specificity as the endogenous LSP1 genes. Constructs having only 66 bp of upstream LSP1β sequences, however, show extremely low levels of CAT expression in tissues and at developmental stages in which LSP1 is not expressed. We discuss the significance of short regions of homology between the DNA upstream of the α and β genes, which lie within the regions identified by the transformation experiments as being required for the cis-regulation of LSP1 synthesis.     

Thermodynamics of solvation for methylproline peptides

We have determined thermodynamic parameters for transfer of N-acetyl,N′-methylamide derivatives of proline and methylprolines from carbon tetrachloride and from chloroform to water. The hydrophilic nature of the diamide model peptides is demonstrated by the negative free energies and enthalpies for transfer. Chloroform solvates the peptides considerably better than carbon tetrachloride. Heats of dilution in carbon tetrachloride arise from disruption of intermolecular peptide–peptide hydrogen bonds. After extrapolation to dilute solution, differences in thermodynamic parameters among the isomeric mono-methylproline peptides are correlated with the population of the intramolecularly hydrogen-bonded C₇ conformer in the nonpolar solvent. However, the thermodynamic parameters aslo reflect differences in solvation due to the proximity of the two peptide groups and the side chain.     

A filtration method for the separation of cell sap from the large-particle fraction of rat liver suspensions, enabling the rapid measurement of nucleotide distributions

A simple method is described that allows a rapid separation of a cell-sap fraction from the large-particle fraction of rat liver suspensions. The method is based on the filtration under suction of liver suspensions through Millipore filters that retain nuclei, mitochondria and some of the endoplasmic-reticulum fraction, but allow quantitative passage of cell sap into a collecting tube. The cell sap may be separated in this manner within 2min of the death of the rat. The method was applied to study the intracellular distribution of ATP and of the nicotinamide-adenine dinucleotides and the results obtained were compared with those obtained after separating the cell sap by a rapid centrifuging procedure. The percentage of total liver ATP in the cell sap was found to be 46% by the filtration method and more than 70% by the centrifuging procedure. Corresponding figures found for the distribution of NADP(+)+NADPH were 40 and 49% respectively.