Cell-cycle-dependent changes in labelling of specific phosphoproteins by the monoclonal antibody MPM-2 in plant cells

The MPM-2 antibody, which recognizes a mitosis-specific phosphorylated epitope, has been used to study cell-cycle-related proteins in partially synchronized cell suspension cultures and root meristem cells. Immunofluorescence revealed that the epitope recognized by MPM-2 is located in the nucleus in interphase cells. In mitotic cells, MPM-2 labels the prophase nucleus, the spindle and some cytoplasmic components. The relative amount of the epitope changes significantly during the cell cycle. Labelling is lowest in G1 and S-phase cells and increases 2–3-fold during G2. Prophase and metaphase show four to five times the labelling of G1 cells. Labelling decreases rapidly after metaphase and is at a very low level by telophase. One- (1-D) and two-dimensional (2-D) immunoblots showed that MPM-2 labels a family of phosphorylated proteins. The labelling shows significant cell cycle dependence. Subfractionation shows at least one of these proteins is a component of the detergent-insoluble cytoskeleton cell fraction. This component is resolved on 2-D immunoblots to two to three spots of slightly different isoelectric point, possibly charge isomers, at a relative molecular mass of approximately 65 kDa. The same spots are labelled by IFA, an antibody against intermediate filament proteins. Another three of the spots at lower relative molecular mass are labelled on 2-D immunoblots of the nuclear matrix fraction.     

The complete amino acid sequences of cytosolic and mitochondrial aspartate aminotransferases from horse heart,and inferences on evolution of the isoenzymes

Summary We report here the complete amino acid sequences of the cytosolic and mitochondrial aspartate aminotransferases from horse heart. The two sequences can be aligned so that 48.1% of the amino acid residues are identical. The sequences have been compared with those of the cytosolic isoenzymes from pig and chicken, the mitochondrial isoenzymes from pig, chicken, rat, and human, and the enzyme fromEscherichia coli. The results suggest that the mammalian cytosolic and mitochondrial isoenzymes have evolved at equal and constant rates whereas the isoenzymes from chicken may have evolved somewhat more slowly. Based on the rate of evolution of the mammalian isoenzymes, the geneduplication event that gave rise to cytosolic and mitochondrial aspartate aminotransferases is estimated to have occurred at least 10⁹ years ago. The cytosolic and mitochondrial isoenzymes are equally related to the enzyme fromE. coli; the prokaryotic and eukaryotic enzymes diverged from one another at least 1.3×10⁹ years ago.     

Production of somatic hybrids of moss by electrofusion

Summary Mixtures of protoplasts of two auxotrophic mutants of Physcomitrella patens, one requiring thiamine (aneurine), the other p-aminobenzoic acid, have been subjected to electrofusion. The protoplasts were aligned in an alternating electric field (500 KHz, 20 V RMS/cm) and induced to fuse by a brief DC pulse (800 V/cm, time constant lms). After culture, first on complete medium and then on selective medium, hybrid plants were obtained at a frequency of 3%. One hybrid with a morphology typical of polyploidy was also observed.Dedicated to Professor Georg Melchers to celebrate his 50-year association with the journal     

Kinetic studies of the uptake of aspartate aminotransferase and malate dehydrogenase into mitochondria in vitro.

Kinetic measurements of the uptake of native mitochondrial aspartate aminotransferase and malate dehydrogenase into mitochondria in vitro were carried out. The uptake of both the enzymes is essentially complete in 1 min and shows saturation characteristics. The rate of uptake of aspartate aminotransferase into mitochondria is decreased by malate dehydrogenase, and vice versa. The inhibition is exerted by isoenzyme remaining outside the mitochondria rather than by isoenzyme that has been imported. The thiol compound beta-mercaptoethanol decreases the rate of uptake of the tested enzymes; inhibition is a result of interaction of beta-mercaptoethanol with the mitochondria and not with the enzymes themselves. The rate of uptake of aspartate aminotransferase is inhibited non-competitively by malate dehydrogenase, but competitively by beta-mercaptoethanol. The rate of uptake of malate dehydrogenase is inhibited non-competitively by aspartate aminotransferase and by beta-mercaptoethanol. beta-Mercaptoethanol prevents the inhibition of the rate of uptake of malate dehydrogenase by aspartate aminotransferase. These results are interpreted in terms of a model system in which the two isoenzymes have separate but interacting binding sites within a receptor in the mitochondrial membrane system.     

Ultrastructural localization of alkaline phosphatase in the cyanobacteriaCoccochloris peniocytis andAnabaena cylindrica

Summary Histochemical techniques applied at the ultrastructural level have established the periplasmic space as the site of cell bound alkaline phosphatase activity inAnabaena cylindrica andCoccochloris peniocytis. For localization of activity unfixed cells were reacted with calcium nitrate, which acts as the initial capture reagent. After this deposition, the cells were suspended in 2% lead nitrate to convert the calcium phosphate to more electron dense lead phosphate. The majority of cell bound activity appeared to be associated with layer 3 of the cell wall. InA. cylindrica a secondary site of cell bound activity appeared to be in the sheath. Placement in a phosphate free medium caused a substantial increase in the enzyme activity ofA. cylindrica while the activity present in log phase cells ofC. peniocytis was similar to that found in phosphate starved cells.C. peniocytis also secretes the enzyme into the surrounding medium.     

Polyploidy‐associated genomic instability in Arabidopsis thaliana

Formation of polyploid organisms by fertilization of unreduced gametes in meiotic mutants is believed to be a common phenomenon in species evolution. However, not well understood is how species in nature generally exist as haploid and diploid organisms in a long evolutionary time while polyploidization must have repeatedly occurred via meiotic mutations. Here, we show that the ploidy increased for two consecutive generations due to unreduced but viable gametes in the Arabidopsis cyclin a1;2‐2 (also named tardy asynchronous meiosis‐2) mutant, but the resultant octaploid plants produced progeny of either the same or reduced ploidy via genomic reductions during meiosis and pollen mitosis. Ploidy reductions through sexual reproduction were also observed in independently generated artificial octaploid and hexaploid Arabidopsis plants. These results demonstrate that octaploid is likely the maximal ploidy produced through sexual reproduction in Arabidopsis. The polyploidy‐associated genomic instability may be a general phenomenon that constrains ploidy levels in species evolution. genesis 48:254–263, 2010. © 2010 Wiley‐Liss, Inc.