Cloning of alkaline phosphatase isozyme gene (iap) of Escherichia coli

In Escherichia coli three major alkaline phosphatase isozymes are formed by molecular conversions depending on physiological conditions. A chromosomal gene, iap, is responsible for alkaline phosphatase isozyme conversion and is assumed to code for a proteolytic enzyme removing the arginine residue(s) from the N-terminal position of alkaline phosphatase subunits. A chromosomal fragment which complemented the Iap^? phenotype was cloned into pBR322 by a shotgun method. Transducing phage λiap was constructed in vitro from the chromosomal fragment containing the iap gene and λtna DNA. The integration site of the phage on chromosome was identified as the iap locus by PI transduction, which meant that the cloned chromosomal DNA contained authentic iap gene.The restriction map of the hybrid plasmid was constructed. Based upon this information, several iap deletion plasmids as well as smaller iup⁺ plasmids were constructed. Analysis of the phenotypes conferred by these plasmids enabled us to locate iap gene within a 2-kb segment of the cloned DNA.The cells carrying the iap⁺ plasmid showed very efficient isozyme conversion even in medium containing arginine, an inhibitor for the isozyme conversion. This indicates overproduction of the iap gene product.     

Auxin-induced changes in the cell wall structure: Changes in the sugar compositions, intrinsic viscosity and molecular weight distributions of matrix polysaccharides of the epicotyl cell wall of Vigna angularis

Rapid effects of indole-3-acetic acid (IAA) on the mechanical properties of cell wall, and sugar compositions, intrinsic viscosity and molecular weight distribution of cell wall polysaccharides were investigated with excised epicotyl segments of Vigna angularis Ohwi et Ohashi cv. Takara.

• 1 IAA caused cell wall loosening as studied by stress-relaxation analysis within 15 min after the IAA application.
• 2 IAA stimulated the decrease in the content of arabinose and galactose in the hemicellulose 1 h after its application. The amounts of other component sugars in the cell wall polysaccharides remained constant during the IAA-induced segment growth.
• 3 The intrinsic viscocity of the pectin increased as early as 30 min after the IAA application. This effect was not prevented when elongation growth of the segment was osmotically suppressed by 0.15 M mannitol.
• 4 Gel permeation chromatography of the pectin on a Sepharose 4 B column demonstrated that IAA caused increase in the mass-average molecular weight of the pectin. Analysis of the sugar compositions of the pectin eluted from the Sepharose 4 B column indicated that IAA increased the molecular weight of the polysaccharides composed of uronic acid, galactose, rhamnose and arabinose. This effect became apparent within 30 min after the IAA application. Furthermore, IAA increased the molecular weight of the pectin when elongation growth of the epicotyl segments was osmotically suppressed by 0.15 M mannitol.
• 5 Hemicellulose of the cell wall chromatographed on a Sepharose CL-4 B column. Analysis of the neutral sugar compositions and the iodine staining property (specific for xyloglucans) of the polysaccharide solution eluted from the column indicated that the hemicellulose consisted of xyloglucans, arabinogalactans and polysaccharides composed of xylose and/or mannose. IAA caused a decrease in the arabinogalactan content and depolymerization of xyloglucans. These IAA effects became apparent within 30 min after the IAA application. These changes occurred even when elongation growth of the epicotyl segments was osmotically suppressed by 0.15 M mannitol.

Polymerization of the pectin, degradation of arabinogalactans and depolymerization of xyloglucans appear to be involved in the mechanism by which IAA induces cell wall loosening and therefore extension growth of cells.     

A Study on the Mechanism of Nodding Initiation of the Flower Stalk in a Poppy, Papaver Rhoeas L.

A study was made to find whether the nodding of the flower stalkin a poppy, Papaver Rhoeas L., immediately after its formationwas triggered by the weight of its flower bud or by positivegeoreaction and the following results were obtained.

1. The direction of the nodding was mostly toward the inclinedside of the stalk, which was opposite the leaf, for apical flowerbuds.
2. If the weight of the flower bud at stage 1 was cancelledbyapplying a load equivalent to the bud weight, the noddingofthe stalk was not initiated.
3. The stalk at stages 1 and2 and the upper part of the stalk(bending zone), as comparedwith the basal part, at later stageswere highly deformableaccording to measurements by the bendingmethod.
4. The cellwall of highly deformable stalks was rich in hemicellulosesand that of the basal part was abundant in pectic substances.

From these results, we concluded that the initiation of thenodding in the flower stalk was caused by the weight of theflower bud and positive geotropic reaction was probably notinvolved. (Received December 22, 1980; Accepted January 26, 1981)     

Chromosome segregation in an asymmetrically dividing bacterium,Caulobacter crescentus

The mode of chromosome segregation in an asymmetrically dividing bacterium, Caulobacter crescentus, was studied by examining the fate of labeled DNA strands. Swarmer cells (one type of Caulobacter daughter cell), in which single strands of DNA had been labeled with [³H]thymidine during the previous round of chromosome replication, were grown synchronously in a non-radioactive medium for two generations. The distribution of radioactivity among the cells was visualized by autoradiography under a phase-contrast microscope. The labeled DNA strands in each cell were found to consist of two conserved units. From this, we propose a model in which the swarmer cell has two identical chromosomes, which are segregated into the progeny swarmer cell and the progeny stalked cell after chromosome replication.     

Radioimmunoassay of myelin basic protein in cerebrospinal fluid and its clinical application to patients with neurological diseases

A double antibody radioimmunoassay for myelin basic protein (BP) was developed that detects BP concentration greater than 0.5 ng/ml in cerebrospinal fluid ( CSF ). By this method, the amount of BP in CSF of 62 patients with various neurological disorders including 9 cases of multiple sclerosis was measured. The amount of BP in CSF obtained from 2 cases of multiple sclerosis in the exacerbation had significantly higher values than that in remission or during the gradual recovering stage. Also two of the 6 patients with myelopathy ( etiology unknown ) and one of the 11 patients with cerebrovascular disease, having acute attack, had significantly high BP values in CSF. The amount of BP in CSF from the other neurological patients showed normal level, compared with that from control patients. The presence of cross-reacting materials with bovine BP in CSF appears to be characteristic of acute myelin sheath destruction on not only patients with multiple sclerosis but also those with myelopathy and cerebrovascular disease.     

Small circular DNA complexes in eucaryotic cells

A small number of eucaryotic cells (100 to 1000 cells) were pressed by mica sheet; then the extruded contents were adsorbed on mica and processed for electron microscopy. In the absence of divalent cation, small polydisperse circular DNA molecules bound to proteins or membrane material were preferentially adsorbed. The small circular DNA complexes have been found in every eucaryotic cell, primary lymphoid tissue cells of bursa and thymus, primary cell lines of retina and liver, and established cultured cell lines of embryonal teratocarcinoma, F9 and PCC3, HeLa and 3T6. Size distribution of these DNA complexes varies, depending on the cell source. The circles less than 1 μm in contour length predominate in cultured cell lines and the larger ones in primary cell lines and cells in situ. Polydisperse covalently closed circular DNAs were recovered from thymus lymphocytes by the conventional dye-CsCl buoyant density method. Their size distribution was similar to that of the small circular DNA complexes detected by the mica-press-adsorption method. They are present in several tens to hundreds of copies per cell representing, at a maximum, 0.02% of the total cellular DNA. The possibility that small circular DNA complexes may result from gene rearrangement as well as from replicon “misfiring” (A. Varshavsky, 1981, Proc. Nat. Acad. Sci. USA 78, 3673–3677) are discussed.     

Trisomy 12p syndrome

Summary The first case of trisomy of probable 12p mosaicism originated de novo is presented. Comparison of the clinical findings of this patient with those of previously described cases of 12p trisomy derived from translocated chromosomes indicates that the symptoms of 12p trisomy are: (1) normal birth weight and physical development, (2) severe psychomotor retardation and generalized hypotonia, (3) peculiarly round face with prominent cheeks, hypertelorism, epicanthus, broad, flat nasal bridge, short nose with anteverted nostrils, large philtrum, broad, prominent lower lip, and (4) poly(syn)dactyly of feet.