Primary structure of hemoglobin β-chain fromColumba livia (gray wild pigeon)

Primary structure of lp6q7514064vv2/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-chain of pigeon is presented. It was determined by amino acid sequence analysis of intact lp6q7514064vv2/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-chain and its peptides obtained by the enzymatic and chemical cleavage. Comparison of amino acid sequence of the chain with other available data shows lp6q7514064vv2/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0"> 14 Ile, lp6q7514064vv2/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">61 Lys, and lp6q7514064vv2/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">113 Ile as residues specific to pigeon. One important replacement at lp6q7514064vv2/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">₁lp6q7514064vv2/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">₁ contact is lp6q7514064vv2/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">55 Metlp6q7514064vv2/xxlarge8594.gif" alt="rarr" align="BASELINE" BORDER="0">Ser.     

Production of thermostable α-galactosidase from thermophilic fungusHumicola sp

The thermophilic fungus,Humicola sp isolated from soil, secreted extracellular lp2571835vt37/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">-galactosidase in a medium cotaining wheat bran extract and yeast extract. Maximum enzyme production was found in a medium containing 5% wheat bran extract as a carbon source and 0.5% beef extract as a carbon and nitrogen source. Enzyme secretion was strongly inhibited by the presence of Cu²⁺, Ni²⁺ and Hg²⁺ (1mM) in the fermentation medium. Production of enzyme under stationary conditions resulted in 10-fold higher activity than under shaking conditions. The temperature range for production of the enzyme was 37° C to 55°C, with maximum activity (5.54 U ml^–1) at 45°C. Optimum pH and temperature for enzyme activity were 5.0 and 60° C respectively. One hundred per cent of the original activity was retained after heating the enzyme at 60°C for 1 h. At 5mM Hg²⁺ strongly inhibited enzyme activity. TheK _m andV _max forp-nitrophenyl-lp2571835vt37/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">-d-galactopyranoside were 60lp2571835vt37/xxlarge956.gif" alt="mgr" align="MIDDLE" BORDER="0">M and 33.6 lp2571835vt37/xxlarge956.gif" alt="mgr" align="MIDDLE" BORDER="0">mol min^–1 mg^–1, respectively, while for raffinose those values were 10.52 mM and 1.8 lp2571835vt37/xxlarge956.gif" alt="mgr" align="MIDDLE" BORDER="0">mol min^–1 mg^–1, respectively.     

The ‘high’ concentrations of enzymes within the chloroplast

In addition to the well documented circumstances associated with ribulose-1,5-bisphosphate carboxylase/oxygenase, calculations suggest that there are several other enzymes of the photosynthetic carbon reduction cycle (i.e. glyceraldehyde-3-phosphate dehydrogenase, aldolase, phosphoribulokinase, transketolase) whose stromal concentrations could readily approach or exceed the concentrations of one or more of their substrates. Such circumstances have extensive ramifications ranging from the alterations of enzyme kinetic behavior and the binding of metabolites to the generation of stromal microenvironments that could facilitate channeling or influence the distribution of metabolites. Consideration of the relative concentrations of all enzymes of the photosynthetic carbon reduction cycle will likely prove essential to a complete understanding of its operation and regulation.     

Incipient forebrain boundaries traced by differential gene expression and fate mapping in the chick neural plate

We correlated available fate maps for the avian neural plate at stages HH4 and HH8 with the progress of local molecular specification, aiming to determine when the molecular specification maps of the primary longitudinal and transversal domains of the anterior forebrain agree with the fate mapped data. To this end, we examined selected gene expression patterns as they normally evolved in whole mounts and sections between HH4 and HH8 (or HH10/11 in some cases), performed novel fate-mapping experiments within the anterior forebrain at HH4 and examined the results at HH8, and correlated grafts with expression of selected gene markers. The data provided new details to the HH4 fate map, and disclosed some genes (e.g., Six3 and Ganf) whose expression domains initially are very extensive and subsequently retract rostralwards. Apart from anteroposterior dynamics, some genes soon became downregulated at the prospective forebrain floor plate, or allowed to identify an early roof plate domain (dorsoventral pattern). Peculiarities of the telencephalon (initial specification and differentiation of pallium versus subpallium) are contemplated. The basic anterior forebrain subdivisions seem to acquire correlated specification and fate mapping patterns around stage HH8.     

Translational control of cellular and viral mRNAs

We are becoming increasingly aware of the role that translational control plays in regulating gene expression in plants. There are now many examples in which specific mechanisms have evolved at the translational level that directly impact the amount of protein produced from an mRNA. All regions of an mRNA, i.e., the 5lp285657/xxlarge8242.gif" alt="prime" align="BASELINE" BORDER="0"> leader, the coding region, and the 3lp285657/xxlarge8242.gif" alt="prime" align="BASELINE" BORDER="0">-untranslated region, have the potential to influence translation. The 5lp285657/xxlarge8242.gif" alt="prime" align="BASELINE" BORDER="0">-terminal cap structure and the poly(A) tail at the 3lp285657/xxlarge8242.gif" alt="prime" align="BASELINE" BORDER="0"> terminus serve as additional elements controlling translation. Many viral mRNAs have evolved alternatives to the cap and poly(A) tail that are functionally equivalent. Nevertheless, for both cellular and viral mRNAs, a co-dependent interaction between the terminal controlling elements appears to be the universal basis for efficient translation.     

Exploring the properties of thermostable Clostridium thermocellum cellulase CelE for the purpose of its expression in plants

The main properties (pH and temperature range, stability, substrate specificity) of the modified cellulase CelE (endo-lp8g657n13g76187/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-1,4-glucanase) from Clostridium thermocellum have been analyzed with the goal of its expression in plants. The modified enzyme is similar to plant cellulases. Deletions in the N-terminus of the enzyme do not affect its biochemical properties. Based on the present investigation, we conclude that the modified lp8g657n13g76187/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-1,4-glucanase CelEM1, when expressed in plants, will be a good model to study the role of cellulases in plants.     

Peroxidase from Catharanthus roseus (L.) G. Don and the biosynthesis of α-3′,4′-anhydrovinblastine: a specific role for a multifunctional enzyme

Summary. We have characterized a basic peroxidase with lp/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">-3lp/xxlarge8242.gif" alt="prime" align="BASELINE" BORDER="0">,4lp/xxlarge8242.gif" alt="prime" align="BASELINE" BORDER="0">-anhydrovinblastine (AVLB) synthase activity, which was purified from Catharanthus roseus leaves. This enzyme was the single peroxidase isoenzyme detected in C. roseus leaves, and the single AVLB synthase activity detected in C. roseus extracts. It was observed that the monomeric substrates of AVLB, vindoline and catharanthine, are both suitable electron donors for the oxidizing intermediates of the basic peroxidase, compounds I and II. Results also showed that the reaction proceeds by a radical-propagated mechanism. Substrate specificity studies of the enzyme revealed that it was also able to oxidize several common peroxidase substrates, indicating a broad range of substrate specificity that is characteristic of class III plant peroxidases. Cytochemical studies showed that the enzyme is localized in C. roseus mesophyll vacuoles, in individual spots at the inner surface of the tonoplast. This particular location suggests a meaningful spatial organization that led to the proposal of a metabolic channeling model for the peroxidase-mediated synthesis of AVLB. The importance of this type of mechanism in the regulation of peroxidase isoenzyme functions in vivo is discussed. In view of the results obtained it is concluded that the basic peroxidase present in C. roseus leaves fulfills all the requirements to be considered as an AVLB synthase, and it is proposed that this specific function of this multifunctional enzyme is determined by metabolic channeling resulting from specific protein–protein interactions.Correspondence and reprints: Institute for Molecular and Cell Biology, University of Porto, rua Campo Alegre 823, 4150-180 Porto, Portugal.Received March 15, 2002; accepted February 4, 2003; published online August 26, 2003     

Analysis And Localization of the Water-Deficit Stress-Induced Gene ( lp3)

LP3 is a water-deficit-induced protein, which is highly homologous to ASR (ABA, stress and ripening) proteins. Homology was found in the C-terminal region of the putative LP3 protein while lower homologies were found in the N-terminal region. The goal of this study was to investigate the function of the LP3 protein and the mechanism of the lp3 promoter in response to water-deficit stress (WDS) and other stresses. In regenerated transgenic tobacco (T₀), expression of -glucuronidase (GUS) from the lp3 promoter-GUS construct was observed in polyethylene glycol (PEG), abscisic acid (ABA), methyl-jasmonate (MeJa), and fluridone (Flu) treatments. GUS expression was not observed following gibberellin (GA₃), 2-methyl-4-dichlorophenoxy acetic acid (2,4-D), silver nitrate, or ethephon (ethylene releasing agent) treatments. Germinated T₁ seedlings containing the lp3 promoter-GUS construct exhibited GUS activity up to 40 days postgermination. Expression could be restored when 5-azacytidine was included in the culture media, indicative of a developmentally regulated silencing mechanism involving methylation. In transgenic tobacco, the LP3 protein localized in the cell nucleus was induced by WDS and appeared to be developmentally regulated. Mailing address of Jau-Tay Wang: 4 Lane 9 Minsheng Rd., Wufeng 413, Taichung, Taiwan. Present address of Jean H. Gould: Department of Forest Science Texas A&M University College Station, Texas 77843-2135, USA; Present address of Veera Padmanabhan: Pioneer HiBred International Inc., 7250 NW 62nd Ave, Johnston, Iowa 50131-0552, USA; Present address of Ronald J. Newton: Department of Biology, East Carolina University, Greenville, North Carolina 27858-4353, USA     

Properties of Bacillus acidocaldarius mutants deficient in ω-cyclohexyl fatty acid biosynthesis

Mutants of the thermoacidophilic Bacillus acidocaldarius, auxotrophic for shikimate or cyclohyxyl-carboxylate, were isolated and characterized. The cyclohexylcarboxylate auxotrophs could be divided by crossfeeding experiments into two groups according to their genetic block. The cyclohexylcarboxylate auxotrophs were deficient in lp462742m0l3v7/xxlarge969.gif" alt="ohgr" align="BASELINE" BORDER="0">-cyclohexyl fatty acid biosynthesis. If the mutants were fed with branched-chain amino acids or short branched-chain fatty acids instead of cyclohexylcarboxylate they form a fatty acid pattern consisting of branched-chain fatty acids. In the high temperature/low pH range the growth yield of cells with this fatty acid pattern is lower as compared to wild type cells or mutants fed with cyclohexylcarboxylate. The same cells are also more sensitive to heat shocks and ethanol. The transport systems for lysine, glutamate and glucose are severely altered by the fatty acid pattern. It was also shown that the density of the lipids containing lp462742m0l3v7/xxlarge969.gif" alt="ohgr" align="BASELINE" BORDER="0">-cyclohexyl fatty acids is higher compared to cells with branched-chain fatty acids. Thus it could be supposed that this alteration influences transport systmes in a direct manner or via energization of the cytoplasmic membrane.