Characterization of β-amylase and its deficiency in various rice cultivars

 β-Amylase deficiency in various cultivars of rice was examined at the molecular level. Using an antibody against β-amylase purified from germinating seeds of rice, we were able to demonstrate the expression and organization of the β-amylase gene in normal and deficient cultivars. Although β-amylase is a starch-hydrolyzing enzyme, as is α-amylase, the β-amylase protein/gene is expressed differently from the α-amylase protein/gene; i.e. (1) β-amylase is synthesized only in aleurone cells, (2) the enzyme production in the embryo-less half-seeds is not under hormonal control. We identified some cultivars of rice that are deficient for β-amylase activity. We present new evidence that synthesis is blocked at the level of mRNA synthesis in the deficient cultivars. The usefulness of β-amylase as a crop trait is also discussed. Received: 8 May 1998 / Accepted: 5 June 1998     

Conformational analysis of biologically active truncated linear analogs of endothelin-1 using NMR and molecular modeling.

Some linear truncated analogs of endothelin-1 display potent agonistic activity at the ET(B) receptor, especially when the side chain of Trp21 is N-formylated. Then, the three-dimensional arrangements of six structurally reduced linear analogs, three formylated and three nonformylated, have been investigated by high resolution NMR spectroscopy and molecular modeling, in order to pinpoint the conformational features related to the biological activity. Two-dimensional double-quantum-filtered correlation spectroscopy (DQFCOSY), total correlation spectroscopy (TOCSY) and nuclear Overhauser enhancement spectroscopy (NOESY) were recorded and analyzed for each molecule. Interspatial distance constraints were derived from the intensity of the NOESY connectivities. The formation of hydrogen bonding was monitored from the temperature dependence of the NH chemical shifts. Molecular models calculated by means of distance geometry, simulated annealing and energy minimization, using the NMR constraints, strongly suggested a global elongated structure for the formylated analogs exhibiting biological activity, and a folded arrangement for the unformylated derivatives. Homology comparisons allowed the identification of a beta-turn-like folding of the C-terminal segment Asp18-Trp21 as a probable key-factor for activity.     

Fetal mouse selenophosphate synthetase 2 (SPS2): biological activities of mutant forms in Escherichia coli.

A novel gene, sps2, detected in mouse embryo at the early stages of development has been identified as an analog of the E. coli selenophosphate synthetase gene. Unlike the E. coli enzyme, the presence of selenocysteine in the mouse enzyme is indicated by a TGA codon in the open reading frame of the cDNA. Using an N-FLAG monoclonal antibody, it was shown that the full length N-FLAG-sps2 gene product was expressed in COS-7 cells. To investigate the biological activity of the sps2 gene product in vivo, the mutated sps2 gene, which contains cysteine in the place of the TGA encoded selenocysteine in the wild type, was expressed in the E. coli selD deficient mutant, MB08. Like the E. coli wild type selD gene, the mutant sps2 gene complemented the selD mutation. However, replacement of Cys with either Ala, Ser, or Thr resulted in a loss of ability to complement the selD mutation. The SPS2-CYS protein expressed in E. coli was purified and its catalytic activity was determined. The Km value for ATP was 0.75 mM and Vmax was 9.23 nmole/min/mg protein. These results confirm that the mouse embryonic sps2 gene encodes an eukaryotic selenophosphate synthetase, and that availability of selenophosphate as a selenium donor compound is widespread.     

Porcine Cloned Embryos Reconstructed with the Cell Nuclei of Tetraploid M-phase Fibroblast Cells Can Restore Normal Diploidy at the Blastocyst Stage

The cell cycle of donor cells as a major factor that affects cloning efficiency remains debatable. G2/M phase cells as a donor can successfully produce cloned animals, but a minimal amount is known regarding nuclear remodeling events. In this study, porcine fetal fibroblasts (PFFs) were carefully synchronized at G1 or M phase as donor cells. Most of the cloned embryos reconstructed from PFFs at G1 (G1-embryos) or M (M-embryos) phase formed a pronucleus-like nucleus (PN) within 6-h post fusion (hpf), but the M-embryos formed PN earlier than the G1-embryos did. Moreover, 77.4% of the M-embryos formed two PNs, whereas the G1-embryos formed a single PN. The rate of extrusion of polar body-like structures by the M-embryos was significantly lower than that extruded by the G1-embryos (26.3% vs. 37.1%, P?相似文献