Contribution to the structural characterization of eucaryotic PSI reaction centre—I. Critical analysis of the polypeptidic composition of different P700 enriched fractions

In thylakoid membranes, several peptides of high MW are present which may interfere with the study of CP1's components. Modifying Cleveland's technique [7] for limited proteolysis, we have characterized the polypeptides found in the 60 kD region. Some may result from incomplete washing of the CF1 while others come from the CP1; indeed, this chlorophyll protein complex, which has a higher MW (above 100 kD), very often undergoes a dissociation into smaller components of about 60 KD MW.Analysis of the protein content of different preparations commonly used to obtain PSI reaction centre enriched fractions has been performed. The and subunits of CF1 are among the main contaminants of most of these preparations. A further purification step is described which can be applied to all these preparations, but numerous peptides are still present in the active fractions. It is most unlikely that all these polypeptides are required for the primary photochemical event, and this emphasizes the necessity to find a new simple method to purify PSI reaction centres.     

The major DNA polymerase in cultured plant cells: Partial purification and correlation with cell multiplication

A DNA polymerase activity was isolated from cells of Oryza sativa L. grown in suspension culture. Molecular mass ( 180,000), optimal requirements for pH (neutral), Mg²⁺ (5–10 mM), Mn²⁺ (1 mM), template preference (activated DNA), lack of activity with native or denatured DNA, and sensitivity to N-ethylmaleimide and ionic strength are similar to those of the vertebrate -polymerase. Like DNA polymerase , the DNA polymerase described in this work is the most abundant in proliferating cells of Oryza sativa L., Parthenocissus tricuspidata (Siebold et Zucc.) Planchon, Acer pseudoplatanus L., and Medicago sativa L. and responds to changes in the rate of cell multiplication. We therefore postulate that this -like DNA polymerase is the replicating enzyme of plant cells.Abbreviations BSA bovine serum albumin - EDTA ethylendiamino-tetracetic acid - DTT dithiothreitol - PTSF p-toluenesulfonyl fluoride     

An enzymatic method for microdetermination of aphidicolin: a promising anticancer drug

We have developed a method, based on the in vitro inhibition of purified human DNA polymerase alpha, the major enzyme of DNA replication, which allows the rapid and accurate determination of pmol amounts of aphidicolin, a promising anticancer drug. The efficacy of this simple method was verified by the determination of aphidicolin in the liver, spleen, blood and urine of mice treated parenterically with the drug. Given its sensitivity and the avoidance of radioactive tracers, this enzymatic method is suitable for the determination of the drug in body fluids and tissue biopsies from living humans. It allows the detection and quantitation of aphidicolin in the presence of inactive metabolite(s) with very similar chemical structure(s) such as those generated by liver microsomal oxidases. The technique will also be useful to monitor the purification of the drug from cultures of Cephalosporium aphidicola.     

Perivitelline space of mammalian oocytes: extracellular matrix of unfertilized oocytes and formation of a cortical granule envelope following fertilization.

Extracellular matrices (ECM) present around unfertilized and fertilized mammalian oocytes were studied ultrastructurally in samples prepared in the presence of ruthenium red to facilitate stabilization of extracellular materials. Unfertilized mouse, hamster, and human oocytes have an ECM comprising granules and filaments in their perivitelline spaces (PVS). This matrix is more abundant in the human than in hamsters and mice. The granule/filament matrix appears identical to the matrix seen between cumulus and corona radiata cells following ruthenium red processing and previously shown to comprise protein and hyaluronic acid. By including ruthenium red during fixation, it is possible to demonstrate the existence of cortical granule exudate in the PVS of fertilized oocytes from hamsters, mice, and humans. Much of the cortical granule exudate is trapped in the PVS and forms a new coat around the fertilized oocyte. This material is particulate when stained with ruthenium red and appears to be uniformly dispersed around the entire oocyte surface. We refer to this new coat as the cortical granule envelope. This envelope is observed in the PVS of all developmental stages up to and including blastocysts in all three species. Following hatching of mouse and hamster blastocysts, the cortical granule envelope is no longer present. Possible functions of this envelope are discussed.     

The molecular evolution of ZFY-related genes in birds and mammals

Summary We report the isolation and nucleotide sequence determination of clones derived from five ZFY-related zinc-finger genes from birds and mammals. These sequences are analyzed with reference to the previously published human genes, ZFX and ZFY, and mouse genes, Zfx, Zfa, Zfy-1, and Zfy-2. The analysis indicates that ZFY-related genes are highly conserved in birds and mammals, and that the rate of nucleotide substitution in the Y-linked genes is not as high as predicted. However, the mouse Zfy-1 and Zfy-2 genes are markedly divergent members of the ZFY gene family; we suggest this relates to X-inactivation of the mouse gene Zfx.     

Molecules,fossils, and the origin of tetrapods

Summary Since the discovery of the coelacanth, Latimeria chalumnae, more than 50 years ago, paleontologists and comparative morphologists have debated whether coelacanths or lungfishes, two groups of lobe-finned fishes, are the closest living relatives of land vertebrates (Tetrapoda). Previously, Meyer and Wilson (1990) determined partial DNA sequences from two conservative mitochondrial genes and found support for a close relationship of lungfishes to tetrapods. We present additional DNA sequences from the 12S rRNA mitochondria gene for three species of the two lineages of lungfishes that were not represented in the first study: Protopterus annectens and Protopterus aethiopicus from Africa and Neoceratodus forsteri (kindly provided by B. Hedges and L. Maxson) from Australia. This extended data set tends to group the two lepidosirenid lungfish lineages (Lepidosiren and Protopterus) with Neoceratodus as their sister group. All lungfishes seem to be more closely related to tetrapods than the coelacanth is. This result appears to rule out the possibility that the coelacanth lineage gave rise to land vertebrates. The common ancestor of lungfishes and tetrapods might have possessed multiple morphological traits that are shared by lungfishes and tetrapods [Meyer and Wilson (1990) listed 14 such traits]. Those traits that seem to link Latimeria and tetrapods are arguably due to convergent evolution or reversals and not to common descent. In this way, the molecular tree facilitates an evolutionary interpretation of the morphological differences among the living forms. We recommended that the extinct groups of lobe-finned fishes be placed onto the molecular tree that has lungfishes and not the coelacanth more closely related to tetrapods. The placement of fossils would help to further interpret the sequence of morphological events and innovations associated with the origin of tetrapods but appears to be problematic because the quality of fossils is not always high enough, and differences among paleontologists in the interpretation of the fossils have stood in the way of a consensus opinion for the branching order among lobefinned fishes. Marshall and Schultze (1992) criticized the morphological analysis presented by Meyer and Wilson (1990) and suggest that 13 of the 14 morphological traits that support the sister group relationship of lungfishes and tetrapods are not shared derived characters. Here we present further alternative viewpoints to the ones of Marshall and Schultze (1992) from the paleontological literature. We argue that all available information (paleontological, neontological, and molecular data) and rigorous cladistic methodology should be used when relating fossils and extant taxa in a phylogenetic framework. Offprint requests to: Axel Meyer     

Molecular Analysis of Plant Defense Responses to Plant Pathogens

A number of inducible plant responses are believed to contribute to disease resistance. These responses include the hypersensitive reaction, phytoalexin synthesis, and the production of chitinase, glucanase, and hydroxyproline-rich glycoproteins. Because of the coordinate induction of these responses, it has been difficult to determine whether they are functional defense responses, and if they are, how they specifically contribute to disease resistance. Recent developments in molecular biology have provided experimental techniques that will reveal the specific contribution of each response to disease resistance. In this paper, we describe a strategy to determine if the hypersensitive reaction is a functional plant defense mechanism.     

The xylanase introns from Cryptococcus albidus are accurately spliced in transgenic tobacco plants

The xylanase gene from Cryptococcus albidus contains seven introns. Genomic and cDNA clones under the control of the CaMV 35S promoter were transferred into tobacco plants using Agrobacterium-mediated cell transformation. The genes were transcribed and the mRNAs were amplified by the polymerase chain reaction using primers on each side of the intron region. About 90% of the amplification products from plants transformed with the genomic clone corresponded to the size of the pre-mRNA (1.2 kb) and 10% represented the spliced product (0.85 kb). The 0.85 kb fragment was cloned and sequenced and the result indicated that the introns from the xylanase gene were accurately spliced by the plant cells.     

Low hydration phase properties of phospholipid mixtures

An experimental investigation of the low hydration phase properties of phospholipid mixtures is described. ²H (D₂O) NMR, X-ray diffraction and differential scanning calorimetry have been used to elucidate the phase properties of mixtures of the mixed chain phospholipids palmitoyloleoylphosphatidylcholine (POPC) and palmitoyloleoylphosphatidylethanolamine (POPE). At 10% hydration pure POPE exhibited a H_II phase above 330 K, a fluid lamellar phase below 315 K, and a minimally hydrated crystalline phase below 300 K. For the 1:1 mixture, the samples exhibited only gel or fluid phases between 270 K and 360 K for hydrations in the range 15% to 30%. Below 15% hydration the mixture exhibited two fluid phases with different repeat spacings, as predicted previously.