Investigating the hows and whys of DNA endoreduplication

Endoreduplication is a form of nuclear polyploidization thatresults in multiple, uniform copies of chromosomes. This processis common in plants and animals, especially in tissues withhigh metabolic activity, and it generally occurs in cells thatare terminally differentiated. In plants, endoreduplicationis well documented in the endosperm and cotyledons of developingseeds, but it also occurs in many tissues throughout the plant.It is thought that endoreduplication provides a mechanism toincrease the level of gene expression, but the function of thisprocess has not been thoroughly investigated. Numerous observationshave been made of endoreduplication, or at least extra cyclesof S-phase, as a consequence of mutations in genes controllingseveral aspects of cell cycle regulation. However, until recentlythere were few studies directed at the molecular mechanismsresponsible for this specialized cell cycle. It is suggestedthat endoreduplication requires nothing more elaborate thana loss of M-phase cyclin-dependent kinase activity and oscillationsin the activity of S-phase cyclin-dependent kinase. Key words: Endoreduplication, gene expression, cell cycle regulation, cyclin-dependent kinase.     

Genetic analysis of amino acid accumulation in opaque-2 maize endosperm

The opaque-2 mutation in maize (Zea mays) is associated with an increased level of free amino acids (FAA) in the mature endosperm. In particular, there is a high concentration of lysine, the most limiting essential amino acid. To investigate the basis for the high-FAA phenotype of opaque-2 maize, we characterized amino acid accumulation during endosperm development of several wild-type and opaque-2 inbreds. Oh545o2 was found to have an exceptionally high level of FAA, in particular those derived from aspartate (Asp) and intermediates of glycolysis. The FAA content in Oh545o2 is 12 times greater than its wild-type counterpart, and three and 10 times greater than in Oh51Ao2 and W64Ao2, respectively. We crossed Oh545o2 to Oh51Ao2 and analyzed the F(2:3) progeny to identify genetic loci linked with the high FAA level in these mutants. Quantitative trait locus mapping identified four significant loci that account for about 46% of the phenotypic variance. One locus on the long arm of chromosome 2 is coincident with genes encoding a monofunctional Asp kinase 2 and a bifunctional Asp kinase-homo-Ser dehydrogenase-2, whereas another locus on the short arm of chromosome 3 is linked with a cytosolic triose phosphate isomerase 4. The results suggest an alternation of amino acid and carbon metabolism leads to overproduction and accumulation of FAA in opaque-2 mutants.     

Enhanced glucose-induced insulin release and endogenous Ca2+ ionophoretic activity in neonatal rat islets following islet-activating protein

Islet-Activating Protein, purified from the culture medium of Bordetella pertussis, enhanced glucose-induced insulin secretion from cultured neonatal rat islets in a calcium dependent manner. This effect was accompanied by an increase in lipid associated Ca2+ ionophoretic activity, as measured by passage of Ca2+ through multilamellar planar membranes containing islet lipid extracts. These findings suggest that the action of IAP in the neonatal islet may be mediated by enhanced entry of extracellular calcium following an effect on membrane lipid composition.     

Optimum conditions for high-resolution gradient analysis

The effect of experimental variables in the photometric scanning of centrifuged sucrose gradients on the apparent resolution was studied. Better resolution was obtained with a flow cell with a comparatively large diameter flow path that was designed for bulk flow than with a flow cell with a comparatively small diameter flow path that was designed for laminar flow. Degradation of resolution caused by an increase in the flow rate was more apparent with the laminar flow cell than with the bulk flow cell. The resolution increased as the illuminated volume of the flow cell decreased but was relatively insensitive to illuminated volume at the lower values tested. Resolution was determined by the number of pea plyribosomes resolved and the shape of their peaks and by the deterioration in shape of peaks given by zones of southern bean mosaic virus during repetitive scanning. It was found that resolution of ribosomal peaks is not a good quantitative method for characterizing instrumental performance.     

Polyribosome degradation as a sensitive assay for endolytic messenger-ribonuclease activity

A method is described whereby picogram amounts of pancreatic RNase can be accurately measured using polyribosomes as substrate. The method involves incubating isolated polysomes in RNase, separating them on gradients, and calculating the number of interribosomal bonds for each profile. The decrease in number of interribosomal bonds is proportional to the amount of enzyme added from 20 to 200 pg of pancreatic RNase per assay when conducted at 2°C for 20 min. Sensitivity can be increased by increasing the temperature or the duration of the incubation.Since the method of analysis of the reaction product is unaffected by exonuclease activity, the assay is specific for endonucleases. It can, therefore be used to assay for the presence of endonucleases as contaminants in preparations of exonucleases and to determine the mode of action of nucleases. The method has been used to measure the amount of soluble and polysome-associated RNase in pea stem tissue.     

Troponin of asynchronous flight muscle

Troponin has been prepared from the asynchronous flight muscle of Lethocerus (water bug) taking special care to prevent proteolysis. The regulatory complex contained tropomyosin and troponin components. The troponin components were Tn-C (18,000 Mr), Tn-T (apparent Mr 53,000) and a heavy component, Tn-H (apparent Mr 80,000). The troponin was tightly bound to tropomyosin and could not be dissociated from it in non-denaturing conditions. A complex of Tn-T, Tn-H and tropomyosin inhibited actomyosin ATPase activity and the inhibition was relieved by Tn-C from vertebrate striated muscle in the presence of Ca2+. However, unlike vertebrate Tn-I, Tn-H by itself was not inhibitory. Monoclonal antibodies were obtained to Tn-T and Tn-H. Antibody to Tn-T was used to screen an expression library of Drosophila cDNA cloned in lambda phage. The sequence of cDNA coding for the protein was determined and hence the amino acid sequence. The Drosophila protein has a sequence similar to that of vertebrate skeletal and cardiac Tn-T. The sequence extends beyond the carboxyl end of the vertebrate sequences, and the last 40 residues are acidic. Part of the sequence of Drosophila Tn-T is homologous to the carboxyl end of the Drosophila myosin light chain MLC-2 and one anti-Tn-T antibody cross-reacted with the light chain. Lethocerus Tn-H is related to the large tropomyosins of Drosophila flight muscle, for which the amino acid sequence is known, since antibodies that recognize this component also recognize the large tropomyosins. Tn-H is easily digested by calpain, suggesting that part of the molecule has an extended configuration. Electron micrographs of negatively stained specimens showed that Lethocerus thin filaments have projections at about 39 nm intervals, which are not seen on thin filaments from vertebrate striated muscle and are probably due to the relatively large troponin complex. Decoration of the thin filaments with myosin subfragment-1 in rigor conditions appeared not to be affected by the troponin. The troponin of asynchronous flight muscle lacks the Tn-I component of vertebrate striated muscle. Tn-H occurs only in the flight muscle and may be involved in the activation of this muscle by stretch.