An improved approach for transformation of plant cells by microinjection: molecular and genetic analysis

A new culture method for the injection of tobacco mesophyll protoplasts has been established. The protoplasts are embedded in a thin layer of alginate and are nourished from the medium in the underlying basislayer. In the alginate layer the protoplasts regenerate to calli at a frequency of up to 80%. Embedded protoplasts can be selected either with 50 mg l⁻¹ kanamycin or 5 mg l⁻¹ paromomycin. Single resistant cells can be recovered from about 10 000 sensitive cells in one alginate layer. Injection of theneo gene (coding for neomycin phosphotransferase II) into protoplast derived single cells in the alginate layer results in kanamycin resistant colonies that can be regenerated to mature plants. These plants express the neomycin phosphotransferase as shown by enzyme activity assay. The integration of the transgene into the plant genome could be proved by Southern hybridization to high molecular weight DNA. With this culture method 100 cells can be injected per hour. Transformation frequencies range from 2 to 20%. In crossing experiments, it was shown that the foreign gene is transmitted to the next generation in a Mendelian fashion.     

Comparison of cytochrome P-450 species which catalyze the hydroxylations of the aromatic ring of estradiol and estradiol 17-sulfate

For identification of microsomal cytochrome P-450 (P-450) enzymes which catalyze 2- or 4-hydroxylations of estrogens in the rat liver, estradiol (E₂) and estradiol 17-sulfate (E₂-17-S) were selected as the substrates and incubated with various kinds of purified P-450 enzymes: PB-1, PB-2, PB-4 and PB-5 obtained from phenobarbital-treated male rats (Sprague-Dawley); MC-1 and MC-5 from 3-methylcholanthrene-treated male rats; and UT-1, UT-2, UT-4 and UT-5 from untreated animals. The reactions were carried out under the P-450-reconstructed system, and the resulting products were determined by HPLC using electrochemical detection. All the enzymes tested were shown to have varying degrees of catalytic activities for 2-hydroxylation of the two substrates; UT-1 and UT-2 had the highest activity. Of the induced P-450 enzymes, PB-2 and MC-1 showed fairly high catalytic activity for 4-hydroxylation of E₂. The P-450 enzymes obtained from the untreated male rats, especially UT-4, showed the highest catalytic activity for 4-hydroxylation of the two substrates. From these results and also from kinetic experiments, the P-450 enzymes which catalyze 2- and 4-hydroxylations of estrogen were considered to be different species. A part of E₂ was converted to such metabolites as estrone and those having a hydroxyl group at positions 6β, 15 or 16, each production of which was estimated to be catalyzed by single or multiple P-450s.     

Intercellular communication between follicular angiotensin receptors and Xenopus laevis oocytes: medication by an inositol 1,4,5- trisphosphate-dependent mechanism

In Xenopus laevis oocytes, activation of angiotensin II (AII) receptors on the surrounding follicular cells sends a signal through gap junctions to elevate cytoplasmic calcium concentration ([Ca2+]i) within the oocyte. The two major candidates for signal transfer through gap junctions into the oocyte during AII receptor stimulation are Ins(1,4,5)P3 and Ca2+. In [3H]inositol-injected follicular oocytes, AII stimulated two- to fourfold increases in phosphoinositide hydrolysis and production of inositol phosphates. Injection of the glycosaminoglycan, heparin, which selectively blocks Ins(1,4,5)P3 receptors, prevented both AII-stimulated and Ins(1,4,5)P3-induced Ca2+ mobilization in Xenopus follicular oocytes but did not affect mobilization of Ca2+ by ionomycin or GTP. These results indicate that the AII-regulated process of gap junction communication between follicular cells and the oocyte operates through an Ins(1,4,5)P3-dependent mechanism rather than through transfer of Ca2+ into the ooplasm and subsequent Ca(2+)-induced Ca2+ release.     

Comparison of histomorphometric values in iliac trabecular bone of beagle dogs raised under different breeding systems.

In order to clarify individual differences in bone metabolism among colony-raised beagle dogs, histomorphometric values of iliac trabecular bone and values of serum biochemical constituents related to bone were examined in 10 and 17 beagle dogs raised, respectively, under our two breeding systems in which differences in factors such as exercise, ultraviolet rays, and mineral content of the diet affect bone metabolism. At the age of 14 months, all dogs were injected with tetracycline hydrochloride and calcein twice for double bone labeling in order to measure dynamic as well as static parameters by bone histomorphometry and the ilium was later biopsied. The measurement on cancellous bone areas of undecalcified iliac sections was performed with a semiautomatic image analyser. Values of total calcium, phosphorus, alkalinephosphatase activity, parathyroid hormone and calcitonin in serum were also determined. The results showed that there were no significant differences between the two groups in histomorphometric values, except for the osteoid volume (p less than 0.05) and osteoid surface/trabecular surface ratio (p less than 0.01) in females, or in serum biochemical constituents, except for alkalinephosphatase activity (p less than 0.001) in males, indicating there were virtually no individual differences in bone metabolism in normal colony raised beagle dogs.     

Biotransformation of tabersonine in cell suspension cultures of Catharanthus roseus.

To investigate the reactions involved in the biosynthesis of vindoline from tabersonine, the bioconversion products formed when the latter compound was fed to cell suspension cultures of Catharanthus roseus were isolated and characterized. Two biotransformation products of tabersonine were isolated and shown to be lochnericine, which is formed by epoxidation of tabersonine at positions 14, 15, and lochnerinine, the 11-methoxylation product of lochnericine. The bioconversion ratio of the main biotransformation product, lochnericine, reached a value of 80.6% within three days.     

Search for MHC-associated genes in human: five new genes centromeric to HLA-DP with yet unknown functions

Taking advantage of five mouse genomic or cDNA probes [KE5(probe 14), KE4 (probe11), KE3 (probe7), KE2 (probe5), and SET] mapped on the H-2K region in mouse, we have identified and localized homologues of these five genes in the human major histocompatibility complex region (HKE5, HKE4, HKE3, HKE2, and HSET, respectively). Cosmid cloning and pulsed field gel electrophoresis analyses indicated that a human homologous gene, HKE5, is located 10 kilobases (kb) centromeric of the 2(XI) collagen (COL11A2) gene followed by HKE4. HKE3, closely linked to HKE2, is located 170 kb centromeric of HKE4. Furthermore, HSET is located 50 kb centromeric of HKE2. This gene organization outside the DP subregion is completely identical to that of the mouse H-2K region centromeric of I-Pb 3, a mouse homologue of the DPB gene, except the lack of genes corresponding to the H-2K and -K2 genes in human.     

Effect of addition of polyethyleneimine on thermal stability and activity of glucose dehydrogenase

When polyethyleneimine (PEI), a water-soluble cationic polymer, was added to solutions of glucose dehydrogenase (GDH) from Bacillus megaterium IWG3 at a molar ratio of PEI to GDH greater than 10, the thermal stability of GDH markedly increased. By addition of PEI, the rate of GDH-catalysed oxidation of -d-glucose increased in a low concentration range of NAD⁺ and NADP⁺ and the Michaelis constants and inhibition constants for both NAD⁺ and NADP⁺ decreased. These results suggested that negatively charged GDH interacts with cationic water-soluble polymers to form conjugates by electrostatic attraction, and also that negatively charged coenzymes are adsorbed by the polymers, resulting in enrichment of the coenzymes in the vicinity of GDH. Addition of PEI was also found to be effective for preventing the denaturation of GDH by acrylamide.Correspondence to: M. Teramoto     

A DBL-homologous region of the yeast CLS4/CDC24 gene product is important for Ca(2+)-modulated bud assembly.

The CLS4/CDC24 is essential for the budding process of the yeast Saccharomyces cerevisiae. Disruption of the CLS4/CDC24 gene is lethal, and expression of the CLS4 product under the control of the GAL1 promoter is sufficient for cellular growth. The CLS4 product is detected in yeast cell lysate with an apparent molecular mass of 93 kD (854 amino acid residues) and shows homology with the human DBL oncogene product. Temperature-sensitive cdc24-1 mutation is located in the N-terminal portion of the protein whereas Ca(2+)-sensitive cls4-1 mutation is present after the DBL-homologous region (amino acid residues 281-518) near the putative Ca(2+)-binding site. Mutations within the DBL-homologous region are responsible for the Ca(2+)-sensitive phenotype. Thus the CLS4 gene product seems to have several functional domains within the molecule essential for bud assembly.