Phytic acid (myo-inositol-1, 2, 3, 4, 5, 6-hexakisphosphate or Ins P 6 ) typically represents approximately 75% to 80% of maize (Zea mays) seed total P. Here we describe the origin, inheritance, and seed phenotype of two non-lethal maize low phytic acid mutants, lpa1-1 and lpa2-1. The loci map to two sites on chromosome 1S. Seed phytic acid P is reduced in these mutants by 50% to 66% but seed total P is unaltered. The decrease in phytic acid P in mature lpa1-1 seeds is accompanied by a corresponding increase in inorganic phosphate (P i ). In mature lpa2-1 seed it is accompanied by increases in P i and at least three other myo-inositol (Ins) phosphates (and/or their respective enantiomers): d-Ins(1,2,4,5,6) P 5 ; d-Ins (1,4,5,6) P 4 ; and d-Ins(1,2,6) P 3 . In both cases the sum of seed P i and Ins phosphates (including phytic acid) is constant and similar to that observed in normal seeds. In both mutants P chemistry appears to be perturbed throughout seed development. Homozygosity for either mutant results in a seed dry weight loss, ranging from 4% to 23%. These results indicate that phytic acid metabolism during seed development is not solely responsible for P homeostasis and indicate that the phytic acid concentration typical of a normal maize seed is not essential to seed function.

No abstract

The 331-kbp chlorovirusParamecium bursariachlorella virus 1 (PBCV-1) genome was resequenced and annotated to correct errors in the original 15-year-old sequence; 40 codons was considered the minimum protein size of an open reading frame. PBCV-1 has 416 predicted protein-encoding sequences and 11 tRNAs. A proteome analysis was also conducted on highly purified PBCV-1 virions using two mass spectrometry-based protocols. The mass spectrometry-derived data were compared to PBCV-1 and its hostChlorella variabilisNC64A predicted proteomes. Combined, these analyses revealed 148 unique virus-encoded proteins associated with the virion (about 35% of the coding capacity of the virus) and 1 host protein. Some of these proteins appear to be structural/architectural, whereas others have enzymatic, chromatin modification, and signal transduction functions. Most (106) of the proteins have no known function or homologs in the existing gene databases except as orthologs with proteins of other chloroviruses, phycodnaviruses, and nuclear-cytoplasmic large DNA viruses. The genes encoding these proteins are dispersed throughout the virus genome, and most are transcribed late or early-late in the infection cycle, which is consistent with virion morphogenesis.

oxidase, where no oxygen was released into the medium from nonenzymatic peroxide decomposition), product formation and peroxide consumption were tightly coupled, and the rate of product formation was identical to that measured under aerobic conditions. Peroxide reactivity was eliminated by a mutation at the Cu H center, which should not be involved in the peroxide shunt. Our data lend support to recent proposals that Cu(II)-superoxide is the active species.

The pH dependence of native PHM and its M314H variant have been studied in detail. For WT PHM the intensity of the Cu-S interaction visible in the Cu(I) EXAFS data is inversely proportional to catalytic activity over the pH range 3 - 8. A previous model based on more limited data was interpreted in terms of two protein conformations involving an inactive met-on form and an active flexible met-off form which derived its catalytic activity from the ability to couple into vibrational modes critical for proton tunneling. The new studies comparing the WT and M314H variant have led to an evolution of this model where the met-on form has been found to be derived from coordination of an additional Met residue, rather than a more rigid conformer of M314 as previously proposed. The catalytic activity of the mutant decreased by 96% due to effects on both kcat and KM but it displayed the same activity/pH profile with a maximum around pH 6. At pH 8, the reduced Cu(I) form gave spectra which could be simulated by replacing the CuM Cu-S(Met) interaction with a Cu-N/O but the data did not unambiguously assign the ligand to the imidazole side chain of H314. At pH 3.5 the EXAFS still showed the presence of a strong Cu-S interaction, establishing that the met-on form observed at low pH in WT cannot be due to a strengthening of the CuM-methionine interaction, but must arise from a different Cu-S interaction. Therefore, lowering the pH causes a conformational change at one of the Cu centers which brings a new S-donor residue into a favorable orientation for coordination to copper and generating an inactive form. Cys coordination is unlikely since all Cys residues in PHM are engaged in disulfide crosslinks. Sequence comparison with the PHM homologues TBM and DBM suggest that M109 (adjacent to the H-site ligands H107 and H108) is the most likely candidate. A model is presented in which H108 protonates with a pKA of 4.6 to generate the inactive low-pH form with CuH coordinated by M109, H107 and H172.

Affinity maturation of antibodies requires a unique process of targeted mutation that allows changes to accumulate in the antibody genes while the rest of the genome is protected from off-target mutations that can be oncogenic. This targeting requires that the same deamination event be repaired either by a mutagenic or a high-fidelity pathway depending on the genomic location. We have previously shown that the BRCT domain of the DNA-damage sensor PARP-1 is required for mutagenic repair occurring in the context of IgH and IgL diversification in the chicken B cell line DT40. Here we show that immunoprecipitation of the BRCT domain of PARP-1 pulls down Ku70 and the DNA–PK complex although the BRCT domain of PARP-1 does not bind DNA, suggesting that this interaction is not DNA dependent. Through sequencing the IgL variable region in PARP-1−/− cells that also lack Ku70 or Lig4, we show that Ku70 or Lig4 deficiency restores GCV to PARP-1−/− cells and conclude that the mechanism by which PARP-1 is promoting mutagenic repair is by inhibiting high-fidelity repair which would otherwise be mediated by Ku70 and Lig4.

The pH dependence of the PHM-catalyzed monooxygenation of dansyl-YVG was studied in two different buffer systems in the pH range of 4-10. The pH-activity profile measured in a sulfonic acid buffer exhibited a maximum at pH 5.8 and became inactive at pH >9. The data could be fit to a model that assumed a protonated unreactive species A, a major reactive species B, and a less reactive species C. B formed in a deprotonation step with pK(a) of 4.6, while C formed and decayed with pK(a)s of 6.8 and 8.2, respectively. The pH dependence was found to be dominated by k(cat), with K(m)(dansyl-YVG) remaining pH-independent over the pH range of 5-8. Acetate-containing buffers shifted the pH maximum to 7.0, and the activity-pH profile could be simulated by formation and decay of a single active species with pK(a)s of 5.8 and 8.3, respectively. The pH-dependent changes in activity could be correlated with a change in the Debye-Waller factor for the Cu-S(met) (M314) component of the X-ray absorption spectrum which underwent a transition from a tightly bound inactive "met-on" form to a conformationally mobile active "met-off" form with a pK(a) which tracked the formation of the active species in both sulfonic acid and acetate-containing buffer systems. The data suggested that the conformational mobility of the bound substrate relative to the copper-superoxo active species is critical to catalysis and further suggested the presence of an accessible vibrational mode coupling Cu-S motion to the H tunneling probability along the Cu-O...H...C coordinate.

No abstract