Plant Transformation by Coinoculation with a Disarmed Agrobacterium tumefaciens Strain and an Escherichia coli Strain Carrying Mobilizable Transgenes

Transformation of Nicotiana tabacum leaf explants was attempted with Escherichia coli as a DNA donor either alone or in combination with Agrobacterium tumefaciens. We constructed E. coli donor strains harboring either the promiscuous IncP-type or IncN-type conjugal transfer system and second plasmids containing the respective origins of transfer and plant-selectable markers. Neither of these conjugation systems was able to stably transform plant cells at detectable levels, even when VirE2 was expressed in the donor cells. However, when an E. coli strain expressing the IncN-type conjugation system was coinoculated with a disarmed A. tumefaciens strain, plant tumors arose at high frequencies. This was caused by a two-step process in which the IncN transfer system mobilized the entire shuttle plasmid from E. coli to the disarmed A. tumefaciens strain, which in turn processed the T-DNA and transferred it to recipient plant cells. The mobilizable plasmid does not require a broad-host-range replication origin for this process to occur, thus reducing its size and genetic complexity. Tumorigenesis efficiency was further enhanced by incubation of the bacterial strains on medium optimized for bacterial conjugation prior to inoculation of leaf explants. These techniques circumvent the need to construct A. tumefaciens strains containing binary vectors and could simplify the creation of transgenic plants.     

Imaging of skeletal muscle function using 18FDG PET: force production, activation, and metabolism

The purpose of thisstudy was to determine whether [¹⁸F]fluorodeoxyglucose(FDG) positron emission tomography (PET) can be used to evaluate muscleforce production, create anatomic images of muscle activity, andresolve the distribution of metabolic activity within exercisingskeletal muscle. Seventeen subjects performed either elbow flexion,elbow extension, or ankle plantar flexion after intravenous injectionof FDG. PET imaging was performed subsequently, and FDG uptake wasmeasured in skeletal muscle for each task. A fivefold increase inresistance during elbow flexion increased FDG uptake in the bicepsbrachii by a factor of 4.9. Differences in relative FDG uptake weredemonstrated as exercise tasks and loads were varied, permittingdifferentiation of active muscles. The intramuscular distribution ofFDG within exercising biceps brachii varied along the transverse andlongitudinal axes of the muscle; coefficients of variation along theseaxes were 0.39 and 0.23, respectively. These findings suggest FDG PETis capable of characterizing task-specific muscle activity andmeasuring intramuscular variations of glucose metabolism withinexercising skeletal muscle.

     

DAX1 mutations map to putative structural domains in a deduced three-dimensional model.

The DAX1 protein is an orphan nuclear hormone receptor based on sequence similarity in the putative ligand-binding domain (LBD). DAX1 mutations result in X-linked adrenal hypoplasia congenita (AHC). Our objective was to identify DAX1 mutations in a series of families, to determine the types of mutations resulting in AHC and to locate single-amino-acid changes in a DAX1 structural model. The 14 new mutations identified among our 17 families with AHC brought the total number of families with AHC to 48 and the number of reported mutations to 42; 1 family showed gonadal mosaicism. These mutations included 23 frameshift, 12 nonsense, and six missense mutations and one single-codon deletion. We mapped the seven single-amino-acid changes to a homology model constructed by use of the three-dimensional crystal structures of the thyroid-hormone receptor and retinoid X receptor alpha. All single-amino-acid changes mapped to the C-terminal half of the DAX1 protein, in the conserved hydrophobic core of the putative LBD, and none affected residues expected to interact directly with a ligand. We conclude that most genetic alterations in DAX1 are frameshift or nonsense mutations and speculate that the codon deletion and missense mutations give insight into the structure and function of DAX1.     

Synaptic organization of the cabbage looper moth ocellus

Summary Chemical and electronic synapses are present in the ocellar synaptic region of the moth, Trichoplusia ni. The chemical synapses all appear to be of the conventional type. Four different chemical synaptic contacts were observed: Receptor cell axons presynaptic to receptor cell axons, receptor cell axons presynaptic to 1st order interneurons, 1st order interneurons presynaptic to receptor cell axons, and 1st order interneurons presynaptic to 1st order interneurons. Two different types of contact made by electronic synapes were observed: Contacts between receptor cell axons and 1st order interneurons, and contacts between 1st order interneurons. The significance of this synaptic arrangement for the generation of on and off responses in the 1st order interneurons is discussed.Supported by NSF Grant BMS 75-07645 and by the VPI & SU Research Division     

The release ofOpius concolor andO. Concolor var.Siculus [Hym.: Braconidae] against the spring generation ofDacus oleae [Dipt.: Trypetidae] on Corfu

Releases ofOpius concolor Szepl. andO. concolor var.siculus Mon. were carried out in the spring to determine if they could be used as a biological control agent against the heavy infestation ofDacus oleae Gmel. which occurs on Corfu at that time. At an initial density of 300–400 parasites per tree the mean parasitism rates of 3rd stage larvae ranged from 30–50% in the 1st week following the release, indicating thatOpius could work well in the spring in the tall trees with large numbers of ripe and heavily infested fruits that are found on Corfu.     

Membrane specializations of dentritic spines and glia in the weaver mouse cerebellum: a freeze-fracture study

Electron microscopy of thin-sectioned and freeze-fractured preparations of the cerebellum of the weaver mouse indicates that the dendritic spines are morphologically identical to those of their normal littermates. The weaver dendritic spines have been characterized as "unattached" since the synaptic input from the parallel fibers is absent (8-10). The entire region around the dendritic spines is taken up by astrocytic processes in the weaver. The outer fracture face of a normal dendritic spine contains aggregations of 10-nm wide particles in the immediate postsynaptic region. Similar particle aggregations occur in the unattached spines of the weaver. Freeze-fracture preparations reveal rectilinear arrays of particles, having a 7-nm center-to-center distance in the glial membranes. Rectilinear arrays are apparently distributed throughout the astrocyte membrane.     

Reproduction of nuclei in Pelomyxa palustris

Light and electron micrographs were made of nuclei in Pelomyxa palustris, a unicellular, multinucleated giant amoeboid organism. We analyzed 1019 pelomyxae and classified their nuclei according to their location in the nuclear cycle. The majority of organisms (56.3%) had interphase nuclei, some of which contained spores of mostly 1-3 μm in diameter. The nuclei had disintegrated in 1.3 % of organisms that appeared to have no nuclei. The remainder (42.4%) had nuclei in the form of spores (1 to 10 μm spheroids) that were in various stages of development and growth. Mitotic figures were seen in some of them, with several chromosome pairs per nucleus. Interchromosomal fibers were seen at anaphase, and newly formed "young" interphase nuclei were observed.     

Identification of Ser424 as the protein kinase A phosphorylation site in CTP synthetase from Saccharomyces cerevisiae.

The URA7-encoded CTP synthetase [EC 6.3.4.2, UTP:ammonia ligase (ADP-forming)] in the yeast Saccharomyces cerevisiae is phosphorylated on a serine residue and stimulated by cAMP-dependent protein kinase (protein kinase A) in vitro. In vivo, the phosphorylation of CTP synthetase is mediated by the RAS/cAMP pathway. In this work, we examined the hypothesis that amino acid residue Ser424 contained in a protein kinase A sequence motif in the URA7-encoded CTP synthetase is the target site for protein kinase A. A CTP synthetase synthetic peptide (SLGRKDSHSA) containing the protein kinase A motif was a substrate (Km = 30 microM) for protein kinase A. This peptide also inhibited (IC50 = 45 microM) the phosphorylation of purified wild-type CTP synthetase by protein kinase A. CTP synthetase with a Ser424 --> Ala (S424A) mutation was constructed by site-directed mutagenesis. The mutated enzyme was not phosphorylated in response to the activation of protein kinase A activity in vivo. Purified S424A mutant CTP synthetase was not phosphorylated and stimulated by protein kinase A. The S424A mutant CTP synthetase had reduced Vmax and elevated Km values for ATP and UTP when compared with the protein kinase A-phosphorylated wild-type enzyme. The specificity constants for ATP and UTP for the S424A mutant CTP synthetase were 4.2- and 2.9-fold lower, respectively, when compared with that of the phosphorylated enzyme. In addition, the S424A mutant enzyme was 2.7-fold more sensitive to CTP product inhibition when compared with the phosphorylated wild-type enzyme. These data indicated that the protein kinase A target site in CTP synthetase was Ser424 and that the phosphorylation of this site played a role in the regulation of CTP synthetase activity.     

Measuring the Coding Potential of Genomic Sequences Througha Combination of Triplet Occurrence Patterns and RNY Preference

The distribution of n-tuplet frequencies is shown to strongly correlate with functionality when examining a genomic sequence in a reading-frame specific manner. The approach described herein applies a coarse-graining procedure, which is able to reveal aspects of triplet usage that are related to protein coding, while at the same time remaining species independent, based on a simple summation of suitable triplet occurrences measures. These quantities are ratios of simple frequencies to suitable mononucleotide-frequency products promoting the incidence of the RNY motif, preferred in the most widely used codons. A significant distinction of coding and noncoding sequences is achieved.Reviewing Editor: Dr. Massimo Di Giulio