Automated sample-preparation technologies in genome sequencing projects.

A robotic workstation system (BioRobot 96OO, QIAGEN) and a 96-well UV spectrophotometer (Spectramax 250, Molecular Devices) were integrated in to the process of high-throughput automated sequencing of double-stranded plasmid DNA templates. An automated 96-well miniprep kit protocol (QIAprep Turbo, QIAGEN) provided high-quality plasmid DNA from shotgun clones. The DNA prepared by this procedure was used to generate more than two mega bases of final sequence data for two genomic projects (Arabidopsis thaliana and Schizosaccharomyces pombe), three thousand expressed sequence tags (ESTs) plus half a mega base of human full-length cDNA clones, and approximately 53,000 single reads for a whole genome shotgun project (Pseudomonas putida).

Convergent pathways for lipochitooligosaccharide and auxin signaling in tobacco cells.

Lipochitooligosaccharides (LCOs) are a novel class of plant growth regulators that activate in tobacco protoplasts the expression of AXI1, a gene implicated in auxin signaling. Transient assays with a chimeric P(AXI)-GUS expression plasmid revealed that the N-octadecenoylated monosaccharide GlcN has all structural requirements for a biological active glycolipid, whereas the inactive N-acylated GalN epimer inhibits LCO action. Specific inhibition of LCO and auxin action shows that both signals are transduced within the tobacco cell via separate pathways that converge at or before AXI1 transcription. Cytokinin is suggested to be a common effector of LCO and auxin signaling. We also show that activation of AXI1 correlates with growth factor-induced cell division.

Auxin inducibility and developmental expression of axi 1: a gene directing auxin independent growth in tobacco protoplasts.

We describe the characterization of axi 1, a tobacco gene isolated by activation T-DNA tagging which apparently plays a role in auxin action. Upon deregulated expression, axi 1 confers on protoplasts the ability to grow in culture not only in the absence of auxin but also in high auxin concentrations where maximal frequencies of cell division are not observed in wild-type protoplasts. In wild-type plants axi 1 is transcribed principally in root tissue. In the tagged plant line, axi 159, axi 1 RNA can be detected in all tissues tested. Freshly isolated wild-type protoplasts require auxin for the accumulation of detectable levels of axi 1 transcript and this precedes maximal levels of cell division. In contrast, axi 1 RNA appears in protoplasts isolated from axi 159 plants in the absence of auxin. axi 1 was localized to 6.2 kb of plant genomic DNA flanking the right T-DNA border sequence. axi 1 is interrupted by nine introns and in tobacco it is a member of a small gene family. Database searching reveals no similarity within the coding region with other genes. Sequences within the fourth intron are similar to those located in the non-coding regions of other plant genes, some of which are known to be auxin inducible. A DNA fragment containing the conserved sequence acts as an auxin responsive element in transient expression assays in wild-type protoplasts and this response is higher in axi 159 protoplasts. This suggests that auxin induced axi 1 expression may be mediated by a region contained within an intron sequence and that the axi 1 product might play a role in this induction.

Activation of a plant gene by T-DNA tagging: auxin-independent growth in vitro.

A transferred DNA (T-DNA) tagging vector with the potential to produce dominant mutations was used with cocultured Agrobacterium tumefaciens and protoplasts to tag genes involved in the action of the plant growth substance auxin. Transgenic calli were selected for their ability to grow in the absence of auxin in the culture media. From one experiment, 12 calli that displayed this phenotype were recovered, of which 11 were able to regenerate into plants. In one plant studied in detail, protoplast division in the absence of auxin genetically cosegregated with a single T-DNA insert. A messenger RNA encoded by a 6.4-kilobase sequence of plant genomic DNA rescued from the mutant is overexpressed relative to untransformed plants. The genomic DNA, as well as a cognate complementary DNA, once transfected into protoplasts promote growth and cell division in vitro in the absence of exogenously added auxin.

[Transmural potential difference of the gastrointestinal tract: its diagnostic value in placing small intestine catheters without roentgen exposure]. / Die transmurale Potentialdifferenz (tPD) des Gastrointestinaltraktes: Ihre diagnostische Nutzung zur röntgenbelastungsfreien Plazierung von Dünndarmsonden.

Functional disorders of the upper gastrointestinal tract frequently require the placement of biopsy tubes into the duodenum. At present, monitoring of correct placement of these tubes usually entails the use of x-rays involving a single or repeated exposures. For accurate placement of duodenal tubes, the difference in TPD between the stomach and the duodenum has been employed as a criterion. Measurement of the TPD is technically simple, can be done at the bedside and obviates the need for using x-rays. Suitable hardware and the procedure are described in detail.