The Uba2 and Ufd1 proteins of Saccharomyces cerevisiae interact with poly(A) polymerase and affect the polyadenylation activity of cell extracts

Poly(A) polymerase is responsible for the addition of the adenylate tail to the 3′ ends of mRNA. Using the two-hybrid system, we have identified two proteins which interact specifically with the Saccharomyces cerevisiae poly(A) polymerase, Pap1. Uba2 is a homolog of ubiquitin-activating (E1) enzymes and Ufd1 is a protein whose function is probably also linked to the ubiquitin-mediated protein degradation pathway. These two proteins interact with Pap1 and with each other, but not with eight other target proteins which were tested in the two-hybrid system. The last 115 amino acids of Uba2, which contains an 82-amino acid region not present in previously characterized E1 enzymes, is sufficient for the interaction with Pap1. Both Uba2 and Ufd1 can be co-immunoprecipitated from extracts with Pap1, confirming in vitro the interaction identified by two-hybrid analysis. Depletion of Uba2 from cells produces extracts which polyadenylate precursor RNA with increased efficiency compared to extracts from nondepleted cells, while depletion of Ufd1 yields extracts which are defective in processing. These two proteins are not components of polyadenylation factors, and instead may have a role in regulating poly(A) polymerase activity. Received: 6 January 1997 / Accepted: 27 February 1997     

Structure,mechanism and evolution of chloroplast transfer RNA processing systems

Chloroplasts of land plants have an active transfer RNA processing system, consisting of an RNase P-like 5 endonuclease, a 3 endonuclease, and a tRNA:CCA nucleotidyltransferase. The specificity of these enzymes resembles more that of their eukaryotic counterparts than that of their cyanobacterial predecessors. Most strikingly, chloroplast RNase P activity almost certainly resides in a protein, rather than in an RNA protein complex as in Bacteria, Archaea, and Eukarya. The chloroplast enzyme may have evolved from a preexisting chloroplast NADP-binding protein. Chloroplast RNase P cleaves pre-tRNA by a reaction mechanism in which at least one of the Mg²⁺ ions utilized by the bacterial ribozyme RNase P is replaced by an amino acid side chain.Abbreviations pre-tRNA precursor to tRNA - pCp cytidine 5, 3-bisphosphate - IC₅₀ inhibitor concentration giving 50% inhibition - GAPDH glyceraldehyde 3-phosphate dehydrogenase     

Photodynamic herbicides: 1. Concept and phenomenology

A new approach to the design of conceptually and phenomenologically new herbicides is described. It involves the joint utilization of tetrapyrrole precursors, such as δ-aminolaevulinic acid (a biodegradable amino acid) and activators of the chlorophyll biosynthetic pathway, such as 2,2′-dipyridyl, in order to induce treated plants to biosynthesize and accumulate massive amounts of tetrapyrrole intermediates of the chlorophyll biosynthetic pathway in the dark (i.e. at night). During the subsequent light period (daylight) the accumulated tetrapyrroles act as potent photodynamic sensitiziers, which in turn result in the death of susceptible plants in a matter of hours. We have therefore proposed to name herbicides that act via this mechanism as photodynamic herbicides, or more pictorially as laser herbicides. From a limited survey of agricultural plant and weed species it appears that photodynamic herbicides exhibit a very pronounced organ, age and species-dependent selectivity. For example, dicotyledonous weeds such as mustard, red-root pigweed, common purslane and lambsquarter are very susceptible while monocotyledonous plants such as corn, wheat, barley and oats are not. The biochemical basis of this selectivity seems to lie, among other things, in the rates of tetrapyrrole turnover and in a differential enhancement by the applied chemicals of the monovinyl and divinyl tetrapyrrole biosynthetic pathways in the various species. A survey of various groups of chemicals (herbicides and other selected biochemicals) that are likely to exhibit photodynamic herbicidal properties is currently under investigation.     

Characterization of a new marine methylotroph

Abstract A methanol-oxidizing bacterium from a marine environment has been isolated and characterized. The bacterium was a Gram-negative rod, capable of growth on methanol and methylamine, but not on multicarbon compounds. It showed a temperature optimum of 30°C, a salt optimum of 0.4% (w/v) and the mol % G + C of its DNA was 46%. Carbon was assimilated via the ribulose monophosphate pathway for formaldehyde fixation during growth on methanol. This bacterium superficially resembled other obligate methylotrophs requiring NaCl reported previously which were designated Methylomonas thalassica . It also appeared similar to many strains of obligate freshwater methylotrophs, except for its NaCl requirement and its lower mol % G + C.     

A peroxisomal glycolate oxidase in the algaMougeotia

Microbodies of the algaMougeotia were isolated in a linear sucrose gradient. The organelles, which moved to the density 1.24 g cm^–3, contained about 70% of the glycolate oxidase (EC 1.1.3.1) found in this alga. The enzyme oxidized glycolate, utilizing either oxygen or 2,6-dichlorophenolindophenol (DCPIP) as the electron acceptor. L-Lactate was an alternate substrate; almost no D-lactate was utilized. In the presence of O₂, a K_m of 415 M was determined for glycolate, whereas the K_m for L-lactate was about 5,000 M. In the presence of DCPIP, lower concentrations of glycolate and L-lactate were sufficient to obtain the highest rates of enzyme activity.Abbreviations DCPIP 2,6-dichlorophenolindophenol Supported by the Deutsche Forschungsgemeinschaft     

Isozymes of the glycolytic and pentose-phosphate pathways in storage tissues of different oilseeds

Isozymes of hexose-phosphate isomerase (HPI; EC 5.3.1.9), pyruvate kinase (PK; EC 2.7.1.40) and 6-phosphogluconate dehydrogenase (6PGDH; EC 1.1.1.44) have been detected in the developing cotyledons of soybean (Glycine max (L.) Merr.), safflower (Carthamnus tinctorius L.) and sunflower (Helianthus annuus L.). In each seed there are two isozymes each of PK and HPI. The isozyme patterns of 6PGDH are more complex: soybean has two forms of the enzyme, safflower three, and sunflower six. In each tissue, at least 25% of the activity of each of the three enzymes is in the plastids. This supports the proposal that the glycolytic and pentose-phosphate pathways are operating in the plastids and that the plastids are the site of long-chain fatty-acid biosynthesis in developing oilseeds.Abbreviations HPI hexose-phosphate isomerase - 6PGDH 6-phosphogluconate dehydrogenase - PK pyruvate kinase     

Enzymes of asparagine synthesis in maize roots

An asparagine synthetase which is active with either glutamine or NH ₄ ⁺ has been found in maize (Zea mays L.) roots. Unlike the enzyme obtained from legume cotyledons, the maize-root enzyme is only slightly more efficient with glutamine (Km, 1.0 mM) than with NH ₄ ⁺ (Km, 2.0–3.0 mM). The activity of this enzyme is higher in the mature root than in the root-tip region, i.e. root cells develop a capacity to make asparagine from glutamine or NH ₄ ⁺ as they mature. -Cyanoalanine synthetase is also present in maize roots. The apparent Km for cysteine is 2.6 mM and for cyanide is 0.57 mM. The enzyme is more active in the root tip than in mature root tissue. Thus, if asparagine were made in the root tip, the cyanide pathway could represent the mechanism of synthesis. It is our contention, however, that this potential is not realized under normal conditions because ¹⁴C-experiments performed previously have indicated a limited availability of both CN and cysteine in the maize root.     

Photosynthetic carbon assimilation in the blue-green alga Coccochloris peniocystis

Abstract. Cells of the blue-green alga Coccochloris peniocystis , grown at air levels of CO₂, were exposed to [^l4C]bicarbonate in the light for periods of 0.5 to 2.0 s followed by exposure to unlabelled bicarbonate for longer periods of time in the light. The kinetics of tracer movement during these pulse-chase experiments demonstrate that the principal mechanism of CO₂ fixation in this alga is the C₃-pathway although an appreciable amount of the C₄ acid aspartate is found as one of the initial products of photosynthesis. Degradation of the labelled aspartate revealed that after 20 s of illumination, over 95% of the radioactivity was located in the β-carboxyl of this C₄ acid. This alga possesses little, if any, capacity for either the enzymatic decarboxylation of C₄ acids or the regeneration of phosphoenolpyruvate (PEP) from pyruvate mediated by the enzyme pyruvate, P_i dikinase. These data further demonstrate the lack of a functional C₄-pathway in this alga.     

Organization of ascending spinal projections in Caiman crocodilus

Summary Ascending spinal projections in the caiman (Caiman crocodilus) were demonstrated with Nauta and Fink-Heimer methods following hemisections of the third spinal segment in a series of twelve animals. These results were compared with earlier data in the literature obtained from a turtle, a snake, and a lizard using the same experimental and histological procedures. The results show remarkable similarities considering that each species represents a different reptilian order with different evolutionary history and habitat. However, the caiman displays several important peculiarities.Although the dorsal funiculus of the caiman contains the largest number of ascending spinal projections of the four species examined, this funiculus has not differentiated into cuneate and gracile fasciculi as is the case in the tegu lizard. The ventro-lateral ascending spinal projections follow a fundamentally similar general morphologic pattern in the four species with only minor variations. The anatomical arrangement in the caiman and tegu lizard appears most similar in the high cervical and the medullary regions; however, this is not the case in midbrain and thalamic regions where considerably more extensive projections are seen in the caiman. In the caiman an extensive spinal connection to the ventro-lateral nucleus of the dorsal thalamus is present; this connection is reminiscent of the mammalian spinal projection to the ventro-basal complex. The caiman has in common with the other three reptilian species a small projection to another dorsal thalamic region that is apparently homologous to the mammalian intralaminar nuclei, which are the destination of the mammalian paleospinothalamic tract.