Characterization of a cDNA encoding cysteine proteinase inhibitor from Chinese cabbage (Brassica campestris L. ssp. pekinensis) flower buds

A cDNA encoding a new phytocystatin isotype named BCPI-1 was isolated from a cDNA library of Chinese cabbage flower buds. The BCPI-1 clone encodes 199 amino acids resulting in a protein much larger than other known phytocystatins. BCPI-1 has an unusually long C-terminus. A BCPI-1 fusion protein expressed in Escherichia coli strongly inhibits the enzymatic activity of papain, a cysteine proteinase. Genomic Southern blot analysis revealed that the BCPI gene is a member of a small multi-gene family in Chinese cabbage. Northern blot analysis showed that it is differentially expressed in the flower bud, leaf and root.     

Effect of abscisic acid, osmolarity and partial desiccation on the development of recalcitrant mango somatic embryos

Inhibition of mango somatic embryo growth was inducedin vitro by treatments for 4 or more weeks with abscisic acid (0–100 M ABA) with and without high osmolarity provided by mannitol (0–10%). High osmolarity and ABA significantly affected somatic embryo length, precocious germination and the production of good quality secondary somatic embryos. High osmolarity also affected root elongation. Abscisic acid was more effective in suppressing growth and development of 0.5 cm-length somatic embryos than smaller somatic embryos. Development beyond the heart stage was significantly inhibited by both ABA and mannitol treatments. The recovery of good quality somatic embryos was enhanced by high levels of ABA (100 M) with and without mannitol (0–5%). Somatic embryos that had been pulsed with ABA were partially desiccated at different relative humidities. Weight loss was affected only by relative humidity; and ABA did not enhance desiccation tolerance.Abbreviations ABA Abscisic acid - 2,4-D 2,4-Dichlorophenoxyacetic acid - MM1 Mango maturation medium - RH Relative humidity     

Biodegradation of the mixtures of 4-chlorophenol and phenol by Comamonas testosteroni CPW301

A 4-chlorophenol (4-CP)-degrading bacterium, strain CPW301, was isolated from soil and identified as Comamonas testosteroni. This strain dechlorinated and degraded 4-CP via a meta-cleavage pathway. CPW301 could also utilize phenol as a carbon and energy source without the accumulation of any metabolites via the same meta-cleavage pathway. When phenol was added as a additional substrate, CPW301 could degrade 4-CP and phenol simultaneously. The addition of phenol greatly accelerated the degradation of 4-CP due to the increased cell mass. The simultaneous degradation of the 4-CP and phenol is useful not only for enhanced cell growth but also for the bioremediation of both compounds, which are normally present in hazardous waste sites as a mixture.     

Development ofgusA reporter gene constructs for cereal transformation: Availability of plant transformation vectors from the CAMBIA Molecular Genetic Resource Service

The use of reporter genes to characterise sequence elements that act to regulate gene expression in transgenic plants has been vital to the development of foreign gene expression strategies for use in cereal transformation. ThegusA locus ofEscherichia coli, which encodes the enzyme-glucuronidase (GUS), is by far the most popular reporter gene used in plant transformation. In this paper we extend the utility of the GUS reporter gene system in cereal transformation by describing and evaluating a number of novel constructs suitable for use in direct gene transfer experiments. These plasmids are all available from the Molecular Genetic Resource Service of the Center for the Application of Molecular Biology to International Agriculture.     

Characterization of adrenergic receptors and related transduction pathways in the liver of the rainbow trout

Epinephrine (EPI) is thought to act by stimulating adenylyl cyclase (ACase) and cAMP production through β-adrenoceptors in the liver of more primitive vertebrates. Recent observations, however, point to an involvement of α₁-adrenoceptors in EPI action, at least in some fish species. The role of the α₁- and β-adrenergic transduction pathways has been investigated in rainbow trout (Oncorhynchus mykiss) hepatic tissue. Radioligand-binding assays with the β-adrenergic antagonist ³H-CGP-12177 using hepatic membranes purified on a discontinuous sucrose gradient confirmed the presence of β-adrenoceptors (K_d0.36 nM, B_max 8.61 fmol · mg⁻¹ protein). We provide the first demonstration of α₁-adrenoceptors in these same membranes; analysis of binding data with the α₁-adrenergic antagonist ³H-prazosin demonstrated a single class of binding sites with a K_dof 15.4 nM and a B_max of 75.2 fmol · mg⁻¹ protein. There is a straight correlation between β-adrenoceptor occupancy, ACase activation and cAMP production. On the contrary, the role of inositol 1,4,5-trisphosphate (IP₃) has to be elucidated; in fact, despite the presence of specific microsomal binding sites for IP₃ (K_d 6.03 nM, B_max 90.2 fmol · mg⁻¹ protein), its cytosolic concentration was not modulated by EPI. On the other hand, we have previously shown in American eel and bullhead hepatocytes that α₁-adrenergic agonists are able to increase intracellular concentrations of IP₃ and Ca²⁺ and to activate glycogenolysis. These data suggest a marked variation in the liver of different fish both in terms of α₁-binding sites affinity and of α₁-adrenoceptor/IP₃/Ca²⁺ transduction systems.     

Expression and secretion of human lipocortin-1 by promoter and signal sequence of STA1 from Saccharomyces diastaticus

Summary For the secretion of human lipocortin-1 (LC-1) in yeast, a expression and secretion vector was constructed by using the promoter and signal sequence of glucoamylase gene (STA1) of Saccharomyces diastaticus. After the cDNA of human LC-1 was ligated with the secretion vector, the resulting hybrid plasmid was transformed into S. diastaticus. When the recombinant S. diastaticus was cultivated in YPD medium, LC-1 was expressed and secreted into the extracellular medium, yielding LC-1 protein at a concentration of 2.5 g/mL.     

Human immunodeficiency virus type 1 Rev is required in vivo for binding of poly(A)-binding protein to Rev-dependent RNAs.

In the absence of Rev or the Rev-responsive element, the Rev-dependent human immunodeficiency virus type 1 (HIV-1) RNAs do not behave as mRNAs; rather, they exhibit nuclear defects in splicing and/or nuclear export and cytoplasmic defects in stability and translation. A translational initiation factor, eIF-5A, has recently been shown to bind specifically to the Rev activation domain. As the binding of poly(A)-binding protein 1 (PAB1) to the poly(A) tail of mRNAs is involved in both the stability and translation of cytoplasmic mRNAs, we investigated whether Rev might influence the association of PAB1 with cytoplasmic HIV-1 RNAs. Antibodies were generated against PAB1. We used these antibodies in an immunoprecipitation assay to detect specific binding of PAB1 to cytoplasmic mRNAs. We found that in the presence of Rev, PAB1 was associated with Rev-dependent and Rev-independent RNAs in the cytoplasm of transfected cells. However, in the absence of functional Rev, we found little or no PAB1 associated with Rev-dependent RNAs. These RNAs were capable of binding PAB1 in vitro. These results demonstrate that HIV-1 RNAs are defective in PAB1 association in the absence of Rev.     

Mutational and crystallographic analyses of the active site residues of the Bacillus circulans xylanase.

Using site-directed mutagenesis we have investigated the catalytic residues in a xylanase from Bacillus circulans. Analysis of the mutants E78D and E172D indicated that mutations in these conserved residues do not grossly alter the structure of the enzyme and that these residues participate in the catalytic mechanism. We have now determined the crystal structure of an enzyme-substrate complex to 108 A resolution using a catalytically incompetent mutant (E172C). In addition to the catalytic residues, Glu 78 and Glu 172, we have identified 2 tyrosine residues, Tyr 69 and Tyr 80, which likely function in substrate binding, and an arginine residue, Arg 112, which plays an important role in the active site of this enzyme. On the basis of our work we would propose that Glu 78 is the nucleophile and that Glu 172 is the acid-base catalyst in the reaction.     

Human xeroderma pigmentosum group G gene encodes a DNA endonuclease.

Because of defective nucleotide excision repair of ultraviolet damaged DNA, xeroderma pigmentosum (XP) patients suffer from a high incidence of skin cancers. Cell fusion studies have identified seven XP complementation groups, A to G. Previous studies have implicated the products of these seven XP genes in the recognition of ultraviolet-induced DNA damage and in incision of the damage-containing DNA strand. Here, we express the XPG-encoded protein in Sf9 insect cells and purify it to homogeneity. We demonstrate that XPG is a single-strand specific DNA endonuclease, thus identifying the catalytic role of the protein in nucleotide excision repair. We suggest that XPG nuclease acts on the single-stranded region created as a result of the combined action of the XPB helicase and XPD helicase at the DNA damage site.