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The volumetric heating of a thin copper target has been studied with time resolved x-ray spectroscopy. The copper target was heated by a plasma produced using the Lawrence Livermore National Laboratory's Compact Multipulse Terawatt (COMET) laser. A variable spaced grating spectrometer coupled to an x-ray streak camera measured soft x-ray emission (800-1550 eV) from the back of the copper target to characterize the bulk heating of the target. Radiation hydrodynamic simulations were modeled in two-dimensions using the HYDRA code. The target conditions calculated by HYDRA were post-processed with the atomic kinetics code CRETIN to generate synthetic emission spectra. A comparison between the experimental and simulated spectra indicates the presence of specific ionization states of copper and the corresponding electron temperatures and ion densities throughout the laser-heated copper target.

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The calibration of the soft x-ray spectral response of a large radius of curvature, high resolution grating spectrometer (HRGS) with a back-illuminated charge-coupled device detector is reported. The instrument is cross-calibrated for the 10-50 Å waveband at the Lawrence Livermore National Laboratory electron beam ion trap (EBIT) x-ray source with the EBIT calorimeter spectrometer. The HRGS instrument is designed for laser-produced plasma experiments and is important for making high dynamic range measurements of line intensities, line shapes, and x-ray sources.

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A 2400 lines/mm variable-spaced grating spectrometer has been used to measure soft x-ray emission (8-22 Å) from laser-produced plasma experiments at Lawrence Livermore National Laboratory's Compact Multipulse Terrawatt (COMET) Laser Facility. The spectrometer was coupled to a Kentech x-ray streak camera to study the temporal evolution of soft x rays emitted from the back of the Mylar and the copper foils irradiated at 10(15) W/cm(2). The instrument demonstrated a resolving power of ∼120 at 19 Å with a time resolution of 31 ps. The time-resolved copper emission spectrum was consistent with a photodiode monitoring the laser temporal pulse shape and indicated that the soft x-ray emission follows the laser heating of the target. The time and spectral resolutions of this diagnostic make it useful for studies of high temperature plasmas.

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We have calibrated the x-ray response of a variable line spaced grating spectrometer, known as the VSG, at the Fusion and Astrophysics Data and Diagnostic Calibration Facility at the Lawrence Livermore National Laboratory (LLNL). The VSG has been developed to diagnose laser produced plasmas, such as those created at the Jupiter Laser Facility and the National Ignition Facility at LLNL and at both the Omega and Omega EP lasers at the University of Rochester's Laboratory for Laser Energetics. The bandwidth of the VSG spans the range of ∼6-60 Å. The calibration results presented here include the VSG's dispersion and quantum efficiency. The dispersion is determined by measuring the x rays emitted from the hydrogenlike and heliumlike ions of carbon, nitrogen, oxygen, neon, and aluminum. The quantum efficiency is calibrated to an accuracy of 30% or better by normalizing the x-ray intensities recorded by the VSG to those simultaneously recorded by an x-ray microcalorimeter spectrometer.

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An inner-shell photoionized x-ray laser pumped by the Linac Coherent Light Source (LCLS) free electron laser has been proposed recently. The measurement of the on-axis 849 eV Ne Kα laser and protection of the x-ray spectrometer from damage require attenuation of the 1 keV LCLS beam. An Al/Cu foil combination is well suited, serving as a low energy bandpass filter below the Cu L-edge at 933 eV. A high resolution grating spectrometer is used to measure the transmission of a candidate filter with an intense laser-produced x-ray backlighter developed at the Lawrence Livermore National Laboratory Jupiter Laser Facility Janus. The methodology and discussion of the observed fine structure above the Cu L-edge will be presented.

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MOTIVATION: The development of an integrated genetic and physical map for the maize genome involves the generation of an enormous amount of data. Managing this data requires a system to aid in genotype scoring for different types of markers coming from both local and remote users. In addition, researchers need an efficient way to interact with genetic mapping software and with data files from automated DNA sequencing. They also need ways to manage primer data for mapping and sequencing and provide views of the integrated physical and genetic map and views of genetic map comparisons. RESULTS: The MMP-LIMS system has been used successfully in a high-throughput mapping environment. The genotypes from 957 SSR, 1023 RFLP, 189 SNP, and 177 InDel markers have been entered and verified via MMP-LIMS. The system is flexible, and can be easily modified to manage data for other species. The software is freely available. AVAILABILITY: To receive a copy of the iMap or cMap software, please fill out the form on our website. The other MMP-LIMS software is freely available at http://www.maizemap.org/bioinformatics.htm.

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MOTIVATION: Because of the unique biological features, a bioinformatic platform for the integrated genetic and physical map of maize is required for storing, integrating, accessing and visualizing the underlying data. RESULTS: The goal of the Maize Mapping Project is to develop a fully integrated genetic and physical map for maize. To display this integrated map, we have developed iMap. iMap has three main components: a relational database (iMapDB), a map graphic browser (iMap Viewer) and a search utility (iMap Search). iMapDB is populated with current genetic and physical map data, describing relationships among genetic loci, molecular markers and bacterial artificial chromosome (BAC) contigs. The database also contains integrated information produced by applying a set of anchoring rules to assign BAC contigs to specific locations on the genetic map. The iMap Viewer and iMap Search functions are combined in the user interface to allow viewing and retrieving many types of genetic and physical map data. The iMap Viewer features side-by-side chromosome-based displays of the genetic map and associated BAC contigs. For each genetic locus, information about marker type or contig can be viewed via pop-up windows that feature links to external data resources. Searches can be conducted for genetic locus, probe or sequence accession number; search results include relevant map positions, anchored BAC contigs and links to the graphical display of relevant chromosomes. iMap can be accessed at http://www.maizemap.org AVAILABILITY: The iMap utility package is available for non-commercial use upon request from the authors.

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Epigenetic regulatory mechanisms heritably alter patterns of gene expression without changes in DNA sequence. Epigenetic states are often correlated with developmentally imposed alterations in genomic DNA methylation and local chromatin structure. Pl-Blotched is a stable epigenetic allele of the maize anthocyanin regulatory gene, purple plant1(pl). Pl-Blotched plants display a variegated pattern of pigmentation that contrasts sharply with the uniformly dark purple pigmentation of plants carrying the dominant Pl-Rhoades allele. Previously, we showed that the lower level of pigmentation in Pl-Blotched is correlated with lower pl mRNA levels and increased DNA methylation at some sites. To explore how DNA methylation, chromatin structure, and developmental stage might contribute to the expression of Pl-Blotched, we used methylation-sensitive restriction enzymes and DNaseI sensitivity assays to compare the methylation status and chromatin structure of Pl-Blotched and Pl-Rhoades at different stages in development. Both alleles exhibit developmentally sensitive changes in methylation. In Pl-Blotched, methylation of two diagnostic HpaII/MspI sites increases progressively, coincident with the juvenile-to-adult transition in growth. In seedlings, the chromatin encompassing the coding region of the gene is less sensitive to DNaseI digestion in Pl-Blotched than in Pl-Rhoades. Developmental maturation from seedling to adult is accompanied by expansion of this closed chromatin domain to include the promoter and downstream flanking sequences. We provide evidence to show that chromatin structure, rather than DNA methylation, is the primary epigenetic determinant for the phenotypic differences between Pl-Blotched and Pl-Rhoades.

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OBJECTIVE: Many young physicians are women, and many are in their childbearing years. Knowing pregnant physicians' status is useful for epidemiological and workforce reasons, yet no studies have compared pregnant with same-age, non-pregnant physicians, an especially appropriate comparison group. STUDY DESIGN: Data from the Women Physicians' Health Study, a national questionnaire-based survey. We compare 87 pregnant with 1148 non-pregnant women physicians, ages 30-40. RESULTS: Pregnant physicians ate more fat, fruits and vegetables, and cheese, but not more dairy than non-pregnant women physicians. While nearly half consumed alcohol, they reported drinking an average of only 0.4 drinks/week, and none smoked. Nearly all took vitamin supplements. Pregnant physicians exercised as much as non-pregnant physicians, and pregnant physicians' self-reported health status was better. Work amount, desire to work less, perceived work control, career satisfaction, and work stress did not significantly differ by pregnancy status. CONCLUSION: The prenatal period may be a time of especially healthy habits and considerable productivity for female physicians.

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OBJECTIVE: The purpose of this study was to evaluate the effect of a staff education program on the documentation skills of registered nurses in the emergency department. DESIGN: A quasiexperimental posttest design was used in this project. METHODS: We conducted classes based on the Emergency Nurses Association's (ENA) Core Curriculum and focused on documentation of neurologic, abdominal, pulmonary, and cardiac assessments. Twenty emergency nurses attended the classes. ED charts were reviewed according to ENA assessment priorities criteria 3 months after the class. Two hundred ED charts completed by registered nurses who attended the classes were compared with 200 ED charts completed by a comparison group of 20 registered nurses who did not attend the classes. RESULTS: Treatment group registered nurses documented significantly more of the ENA criteria in each system area than did the registered nurses in the comparison group. CONCLUSION: Findings from this study support the importance of continued staff education in the improvement of emergency nurses' documentation of patient assessment.

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The maize pl locus encodes a transcriptional activator of anthocyanin biosynthetic genes. The Pl-Rhoades (Pl-Rh) allele confers robust purple anthocyanin pigment in several tissues. Spontaneous derivatives of Pl-Rh, termed Pl'-mahogany (Pl'-mah), arise that confer reduced pigment and are meiotically heritable. These derivatives influence other Pl-Rh alleles such that only Pl'-mah alleles are transmitted form a Pl-Rh/Pl'mah heterozygote. Genetic crosses establish that chromosomal segregation distortion does not explain this exclusive transmission and suggest that Pl-Rh invariably changes to Pl'-mah when exposed to Pl'-mah. Such behavior is a hallmark of paramutation. Cosegregation experiments demonstrate that this paramutagenic activity is genetically linked to the pl locus. By visually quantifying pl action through successive crosses, we find that phenotypic expression is inversely related to paramutation at two other maize loci, b and r. Previous analysis of b and r paramutation revealed extensive differences and led to suggestions of distinct molecular mechanisms. Consideration of the common features of all three systems reinvigorates the interpretation that the mechanistic processes of these three allelic interactions are similar.

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Genetic studies in maize have identified several regulatory genes that control the tissue-specific synthesis of purple anthocyanin pigments in the plant. c1 regulates pigmentation in the aleurone layer of the kernel, whereas pigmentation in the vegetative and floral tissues of the plant body depends on pl. c1 encodes a protein with the structural features of eukaryotic transcription factors and functions to control the accumulation of transcripts for the anthocyanin biosynthetic genes. Previous genetic and molecular observations have prompted the hypothesis that c1 and pl are functionally duplicate, in that they control the same set of anthocyanin structural genes but in distinct parts of the plant. Here, we show that this proposed functional similarity is reflected by DNA sequence homology between c1 and pl. Using a c1 DNA fragment as a hybridization probe, genomic and cDNA clones for pl were isolated. Comparison of pl and c1 cDNA sequences revealed that the genes encode proteins with 90% or more amino acid identity in the amino- and carboxyl-terminal domains that are known to be important for the regulatory function of the C1 protein. Consistent with the idea that the pl gene product also acts as a transcriptional activator is our finding that a functional pl allele is required for the transcription of at least three structural genes in the anthocyanin biosynthetic pathway.

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The pl gene encodes a regulatory protein that controls the transcription of a number of structural genes of the anthocyanin biosynthetic pathway in maize. pl alleles have been classified phenotypically into two categories: dominant (Pl) alleles lead to intense, light-independent pigmentation in vegetative and floral organs of the plant; recessive "sun-red" alleles (pl) lead to light-dependent red pigmentation in which only tissues exposed to light become pigmented. Based on these observations, two alternate pathways leading to anthocyanin synthesis in the plant have been proposed: one requiring light and the other bypassing the light requirement through the action of Pl. To evaluate this hypothesis, we have analyzed light-independent and light-dependent alleles of pl. Sequence analysis revealed that the two types of alleles have very distinct promoters but have the capacity to encode very similar proteins. The protein encoded by one recessive allele was shown to be functional in transient assays. Measurements of husk mRNA levels by quantitative polymerase chain reaction showed that sun-red pl alleles are expressed at much lower levels than a Pl allele, but their expression is increased approximately sixfold by exposure to light. These results lead to the conclusion that the sun-red pl alleles are not null; instead, they synthesize functional mRNA and protein. We propose that the light-dependent pigmentation observed in pl plants is the result of a threshold effect in which light exposure boosts pl mRNA expression past a crucial level necessary to generate sufficient PL protein molecules to activate transcription of the anthocyanin structural genes.

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Anthocyanins are purple pigments that can be produced in virtually all parts of the maize plant. The spatial distribution of anthocyanin synthesis is dictated by the organ-specific expression of a few regulatory genes that control the transcription of the structural genes. The regulatory genes are grouped into families based on functional identity and DNA sequence similarity. The C1/Pl gene family consists of C1, which controls pigmentation of the kernel, and Pl, which controls pigmentation of the vegetative and floral organs. We have determined the relationship of another gene, Blotched (Bh), to the C1 gene family. Bh was originally described as a gene that conditions blotches of pigmentation in kernels homozygous for recessive c1, suggesting that Bh could functionally replace C1 in the kernel. Our genetic and molecular analyses indicate that Bh is an allele of Pl, that we designate Pl-Bh. Pl-Bh differs from wild-type Pl alleles in two respects. In contrast to the uniform pigmentation observed in plants carrying Pl, the pattern of pigmentation in plants carrying Pl-Bh is variegated. Pl-Bh leads to variegated pigmentation in virtually all tissues of the plant, including the kernel, an organ not pigmented by other Pl alleles. To address the molecular basis for the unusual pattern of expression of Pl-Bh, we cloned and sequenced the gene. The nucleotide sequence of Pl-Bh showed only a single base-pair difference from that of Pl. However, genomic DNA sequences associated with Pl-Bh were found to be hypermethylated relative to the same sequences around the wild-type Pl allele. The methylation was inversely correlated with Pl mRNA levels in variegated plant tissues. Thus, we conclude that DNA methylation may play a role in regulating Pl-Bh expression.

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orange pericarp (orp) is a seedling lethal mutant of maize caused by mutations in the duplicate unlinked recessive loci orp1 and orp2. Mutant seedlings accumulate two tryptophan precursors, anthranilate and indole, suggesting a block in tryptophan biosynthesis. Results from feeding studies and enzyme assays indicate that the orp mutant is defective in tryptophan synthase beta activity. Thus, orp is one of only a few amino acid auxotrophic mutants to be characterized in plants. Two genes encoding tryptophan synthase beta were isolated from maize and sequenced. Both genes encode polypeptides with high homology to tryptophan synthase beta enzymes from other organisms. The cloned genes were mapped by restriction fragment length polymorphism analysis to approximately the same chromosomal locations as the genetically mapped factors orp1 and orp2. RNA analysis indicates that both genes are expressed in all tissues examined from normal plants. Together, the biochemical, genetic, and molecular data verify the identity of orp1 and orp2 as duplicate structural genes for the beta subunit of tryptophan synthase.

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The B, R, C1, and Pl genes regulating the maize anthocyanin pigment biosynthetic pathway encode tissue-specific transcriptional activators. B and R are functionally duplicate genes that encode proteins with the basic-helix-loop-helix (b-HLH) motif found in Myc proteins. C1 and Pl encode functionally duplicate proteins with homology to the DNA-binding domain of Myb proteins. A member of the b-HLH family (B or R) and a member of the myb family (C1 or Pl) are both required for anthocyanin pigmentation. Transient assays in maize and yeast were used to analyze the functional domains of the B protein and its interaction with C1. The results of these studies demonstrate that the b-HLH domain of B and most of its carboxyl terminus can be deleted with only a partial loss of B-protein function. In contrast, relatively small deletions within the B amino-terminal-coding sequence resulted in no trans-activation. Analysis of fusion constructs encoding the DNA-binding domain of yeast GAL4 and portions of B failed to reveal a transcriptional activation domain in the B protein. However, an amino-terminal domain of B was found to recruit a transcriptional activation domain by an interaction with C1. Formation of this complex resulted in the activation of a synthetic promoter containing GAL4 recognition sites, demonstrating that this interaction does not require the normal target promoters for B and C1. B and C1 fusions with yeast GAL4 DNA-binding and transcriptional activation domains were also found to interact when synthesized and assayed in yeast. The domains responsible for this interaction map to a region that contains the Myb homologous repeats of the C1 protein and to the amino terminus of the B protein, which does not contain the b-HLH motif. These studies suggest that the regulation of the maize anthocyanin pigmentation pathway involves a direct interaction between members of two distinct classes of transcriptional activators.

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Genes encoding fusions between the maize regulatory protein C1 and the yeast transcriptional activator GAL4 and mutant C1 proteins were assayed for their ability to trans-activate anthocyanin biosynthetic genes in intact maize tissues. The putative DNA-binding region of C1 fused to the transcriptional activation domain of GAL4 activated transcription of anthocyanin structural gene promoters in c1 aleurones, c1 Rscm2 embryos, and c1 r embryogenic callus. Cells receiving these constructs accumulated purple anthocyanin pigments. The C1 acidic region fused to the GAL4 DNA-binding domain activated transcription of a GAL4-regulated promoter. An internal deletion of C1 also induced pigmentation; however, frameshifts in either the amino-terminal basic or carboxy-terminal acidic region blocked trans-activation, and the latter generated a dominant inhibitory protein. Fusion constructs between the wild-type C1 cDNA and the dominant inhibitor allele C1-I cDNA were used to identify the amino acid changes in C1 responsible for the C1-I inhibitory phenotype. Results from these studies establish that amino acids within the myb-homologous domain are critical for transcriptional activation.

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The C1, B and R genes regulating the maize anthocyanin biosynthetic pathway encode tissue-specific regulatory proteins with similarities to transcriptional activators. The C1 and R regulatory genes are usually responsible for pigmentation of seed tissues, and the B-Peru allele of B, but not the B-I allele, can substitute for R function in the seed. In this study, members of the B family of regulatory genes were delivered to intact maize tissues by high velocity microprojectiles. In colorless r aleurones or embryos, the introduction of the B-Peru genomic clone or the expressed cDNAs of B-Peru or B-I resulted in anthocyanin-producing cells. Luciferase produced from the Bronze1 anthocyanin structural gene promoter was induced 100-fold when co-introduced with the expressed B-Peru or B-I cDNAs. This quantitative transactivation assay demonstrates that the proteins encoded by these two B alleles are equally able to transactivate the Bronze1 promoter. Analogous results were obtained using embryogenic callus cells. These observations suggest that one major contribution towards tissue-specific anthocyanin synthesis controlled by the various alleles of the B and R genes is the differential expression of functionally similar proteins.

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Transposon tagging has become the method of choice for isolating genes whose products are in low abundance. We have recently used the transposable element Spm to tag and clone maize regulatory loci. Our choice of Spm was dictated by several factors: The frequency of transposition of Spm is high enough to obtain detectable transposition events, into loci affecting kernel traits, in populations of less than 10(6) seed. Although the copy number of Spm is high in the maize genome, insertions into the gene of interest can be distinguished from other Spm copies by digesting DNAs from segregating populations with methyl-sensitive restriction enzymes, and hybridizing with Spm-specific probes. Since all members of the Spm family thus far examined share DNA homology, hybridization with appropriate probes allows detection of insertions of both autonomous and defective elements. Thus, if a mutable allele can be shown to be under Spm control, one can be reasonably confident of successfully cloning that allele.

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