A PCR-based test for species-specific determination of heat treatment conditions of animal meals as an effective prophylactic method for bovine spongiform encephalopathy.

Efficiency of animal waste sterilization prescribed by the European Union and Switzerland was verified using a pork-based ELISA and two PCR assays (tRNA(Glu)/cytochrome b specific for vertebrates; bovine species-specific cytochrome b mitochondrial genome). A total of 204 samples of feedingstuffs were analysed including reference materials subjected to known heat treatments. Both ELISA and PCR assays were able to detect poorly heat-treated feedingstuffs if there was enough pork-based material present. The proposed species-specific PCR test, however, showed a higher sensitivity and specificity as it specifically detected bovine material. Nevertheless, the PCR assay will not detect bovine material in properly heat-treated feeds as the DNA is too fragmented. It is, however, very useful as a rapid, sensitive, and specific method for the routine screening of animal meals with regard to prophylaxis of BSE.

Insensitivity of barley endosperm ADP-glucose pyrophosphorylase to 3-phosphoglycerate and orthophosphate regulation.

Crude extracts of starchy endosperm from barley (Hordeum vulgare cv Bomi) contained high pyrophosphorolytic activity (up to 0.5 mumol of glucose-1-P formed min-1 mg-1 of protein) of ADP-glucose pyrophosphorylase (AGP) when assayed in the absence of 3-phosphoglycerate (3-PGA). This high activity was observed regardless of whether AGP had been extracted in the presence or absence of various protease inhibitors or other protectants. Western blot analysis using antibodies specific for either the small or large subunit of the enzyme demonstrated that the large, 60-kD subunit was prone to proteolysis in crude extracts, with a half-time of degradation at 4 degrees C (from 60 to 53 to 51 kD) on the order of minutes. The presence of high concentrations of protease inhibitors decreased, but did not prevent this proteolysis. The small, 51-kD subunit of barley endosperm AGP was relatively resistant to proteolysis, both in the presence or absence of protease inhibitors. For the crude, nonproteolyzed enzyme, 3-PGA acted as a weak activator of the ADP-glucose synthetic reaction (about 25% activation), whereas in the reverse reaction (pyrophosphorolysis) it served as an inhibitor rather than an activator. For both the synthetic and pyrophosphorolytic reactions, inorganic phosphate (Pi) acted as a weak competitive or mixed inhibitor of AGP. The relative insensitivity to 3-PGA/Pi regulation has been observed with both the nonproteolyzed crude enzyme and partially purified (over 60-fold) AGP, the latter characterized by two bands for the large subunit (molecular masses of 53 and 51 kD) and one band for the small subunit (51 kD). Addition of 3-PGA to assays of the partially purified, proteolyzed enzyme had little or no effect on the Km values of all substrates of AGP, but it reduced the Hill coefficient for ATP (from 2.1 to 1.0). These findings are discussed with respect to previous reports on the structure and regulation of higher plant AGP.

The beta-mannanase from "Caldocellum saccharolyticum" is part of a multidomain enzyme.

The complete sequence of a beta-mannanase gene from an anaerobic extreme thermophile was determined, and it shows that the expressed protein consists of two catalytic domains and two binding domains separated by spacer regions rich in proline and threonine residues. The amino-terminal catalytic domain has beta-mannanase activity, and the carboxy-terminal domain acts as an endoglucanase. Neither domain shows homology with any other cellulase or hemicellulase sequence at the nucleic acid or protein level.

PCR amplification and sequences of cDNA clones for the small and large subunits of ADP-glucose pyrophosphorylase from barley tissues.

Several cDNAs encoding the small and large subunit of ADP-glucose pyrophosphorylase (AGP) were isolated from total RNA of the starchy endosperm, roots and leaves of barley by polymerase chain reaction (PCR). Sets of degenerate oligonucleotide primers, based on previously published conserved amino acid sequences of plant AGP, were used for synthesis and amplification of the cDNAs. For either the endosperm, roots and leaves, the restriction analysis of PCR products (ca. 550 nucleotides each) has revealed heterogeneity, suggesting presence of three transcripts for AGP in the endosperm and roots, and up to two AGP transcripts in the leaf tissue. Based on the derived amino acid sequences, two clones from the endosperm, beps and bepl, were identified as coding for the small and large subunit of AGP, respectively, while a leaf transcript (blpl) encoded the putative large subunit of AGP. There was about 50% identity between the endosperm clones, and both of them were about 60% identical to the leaf cDNA. Northern blot analysis has indicated that beps and bepl are expressed in both the endosperm and roots, while blpl is detectable only in leaves. Application of the PCR technique in studies on gene structure and gene expression of plant AGP is discussed.

In vitro mutagenesis of a xylanase from the extreme thermophile Caldocellum saccharolyticum.

Six mutant xylanases were obtained by in vitro mutagenesis of a xylanase gene from the extremely thermophilic bacterium Caldocellum saccharolyticum. The temperature stability of all enzymes was affected by mutation to various degrees and one of the xylanases had an altered temperature optimum. The mutations had no effect on the pH optimum. The C. saccharolyticum xylanase showed strong homology to several thermophilic and mesophilic xylanases, and comparison of primary sequences allowed the localization of probable active sites and residues involved in thermostability.

Plant ADP-glucose pyrophosphorylase--recent advances and biotechnological perspectives (a review).

Recent advances in studies on plant ADP-glucose pyrophosphorylase (AGP), the key enzyme of starch biosynthesis, are presented. AGP constitutes the first committed and highly regulated step of starch synthesis in all plant tissues. The importance of AGP in carbohydrate metabolism and several of its features, such as potent regulation by cellular effectors (3-phosphoglycerate and Pi), an unusual two subunit-types structure, tissue-specific and developmentally-regulated expression, and presence of the AGP-deficient mutants, make it an attractive, but complex, target for biotechnological manipulations. Some strategies for future research on AGP are discussed.

Cloning, sequence analysis, and expression in Escherichia coli of a gene coding for a beta-mannanase from the extremely thermophilic bacterium "Caldocellum saccharolyticum".

A lambda recombinant phage expressing beta-mannanase activity in Escherichia coli has been isolated from a genomic library of the extremely thermophilic anaerobe "Caldocellum saccharolyticum." The gene was cloned into pBR322 on a 5-kb BamHI fragment, and its location was obtained by deletion analysis. The sequence of a 2.1-kb fragment containing the mannanase gene has been determined. One open reading frame was found which could code for a protein of Mr 38,904. The mannanase gene (manA) was overexpressed in E. coli by cloning the gene downstream from the lacZ promoter of pUC18. The enzyme was most active at pH 6 and 80 degrees C and degraded locust bean gum, guar gum, Pinus radiata glucomannan, and konjak glucomannan. The noncoding region downstream from the mannanase gene showed strong homology to celB, a gene coding for a cellulase from the same organism, suggesting that the manA gene might have been inserted into its present position on the "C. saccharolyticum" genome by homologous recombination.

Overproduction of an acetylxylan esterase from the extreme thermophile "Caldocellum saccharolyticum" in Escherichia coli.

The xynC gene coding for an acetylxylan esterase from the extreme thermophile "Caldocellum saccharolyticum" was overexpressed in Escherichia coli strain RR28 by cloning the gene downstream from the lacZ promoter region of pUC18 (pNZ1447) or downstream from the temperature-inducible lambda pRpL promoters of pJLA602 (pNZ1600). The protein formed high molecular weight aggregates in induced cells of RR28/pNZ1600 but not in RR28/pNZ1447. The enzyme constituted up to 10% of the total cell protein and was located in the cytoplasmic fraction of RR28/pNZ1447. The acetyl esterase was most active at pH 6.0 and 70-75 degrees C with a half-life of 64 h at 70 degrees C and 30 h at 80 degrees C, respectively.

Xylanase from the extremely thermophilic bacterium "Caldocellum saccharolyticum": overexpression of the gene in Escherichia coli and characterization of the gene product.

A xylanase encoded by the xynA gene of the extreme thermophile "Caldocellum saccharolyticum" was overexpressed in Escherichia coli by cloning the gene downstream from the temperature-inducible lambda pR and pL promoters of the expression vector pJLA602. Induction of up to 55 times was obtained by growing the cells at 42 degrees C, and the xylanase made up to 20% of the whole-cell protein content. The enzyme was located in the cytoplasmic fraction in E. coli. The temperature and pH optima were determined to be 70 degrees C and pH 5.5 to 6, respectively. The xylanase was stable for at least 72 h if incubated at 60 degrees C, with half-lives of 8 to 9 h at 70 degrees C and 2 to 3 min at 80 degrees C. The enzyme had high activity on xylan and ortho-nitrophenyl beta-D-xylopyranoside and some activity on carboxymethyl cellulose and para-nitrophenyl beta-D-cellobioside. The gene was probably expressed from its own promoter in E. coli. Translation of the xylanase overproduced in E. coli seemed to initiate at a GTG codon and not at an ATG codon as previously determined.

Cloning, sequence analysis, and expression of genes encoding xylan-degrading enzymes from the thermophile "Caldocellum saccharolyticum".

A lambda recombinant bacteriophage coding for xylanase and beta-xylosidase activity has been isolated from a genomic library of the extremely thermophilic anaerobe "Caldocellum saccharolyticum." Partial Sau3AI fragments of the lambda recombinant DNA were ligated into pBR322. A recombinant plasmid with an insertion of ca. 7 kilobases of thermophilic DNA expressing both enzymatic activities was isolated. The location of the genes has been established by analyzing deletion derivatives, and the DNA sequence of 6.067 kilobases of the insert has been determined. Five open reading frames (ORFs) were found, one of which (ORF1; Mr 40,455) appears to code for a xylanase (XynA) which also acts on o-nitrophenyl-beta-D-xylopyranoside. Another, ORF5 (Mr 56,365), codes for a beta-xylosidase (XynB). The xynA gene product shows significant homology with the xylanases from the alkalophilic Bacillus sp. strain C125 and Clostridium thermocellum.

The arc operon for anaerobic arginine catabolism in Pseudomonas aeruginosa contains an additional gene, arcD, encoding a membrane protein.

The arginine deiminase (ADI) pathway in Pseudomonas aeruginosa serves to generate ATP. The three enzymes involved, ADI, catabolic ornithine carbamoyltransferase and carbamate kinase, are induced by oxygen limitation and encoded by the contiguous arcABC genes. A 1.5-kb region upstream from arcABC was sequenced and found to contain an open reading frame, arcD, coding for a hydrophobic polypeptide of 52 kDa. The content and distribution of hydrophobic amino acids suggest that the arcD gene product may be a transmembrane protein. When arcD was fused to an Escherichia coli promoter, the ArcD protein was synthesized in E. coli maxicells and detected in the membrane fraction. In sodium dodecyl sulfate-polyacrylamide-gel electrophoresis the ArcD protein migrated like a 32-kDa protein; such anomalous electrophoretic mobility is known for other highly hydrophobic proteins. Mutations in arcD rendered the cells unable to utilize extracellular arginine as an energy source. Since anaerobic arginine consumption and ornithine release are coupled in P. aeruginosa, it is proposed that arcD specifies an arginine: ornithine antiporter or a part thereof. Insertions of IS21 or Tn1725 in arcD had a strong polar effect on the expression of the arcAB enzymes, indicating that the arc genes are organized as an arcDABC operon.

The arcABC operon required for fermentative growth of Pseudomonas aeruginosa on arginine: Tn5-751-assisted cloning and localization of structural genes.

Pseudomonas aeruginosa is able to utilize L-arginine as the energy source for growth under anaerobic, nitrate-free conditions. Mutations in the chromosomal arcABC gene cluster specifying the inducible arginine deiminase pathway enzymes abolish fermentative growth on arginine. From two different arc::Tn5-751 insertion mutants of P. aeruginosa recombinant plasmids have been derived which carry a resistance marker of transposon Tn5-751 plus flanking parts of the arc region. These recombinant plasmids served to reconstruct in vitro the functional arcABC cluster on a 5.6 kb fragment, which was inserted into the broad-host-range vector pKT240. In P. aeruginosa this 5.6 kb segment complemented arcABC mutations in trans and contained the control region necessary in cis for arc enzyme induction by oxygen limitation and arginine. The results of subcloning experiments and transcriptional lacZ fusions, the polarity of transposon insertions and the effect of external promoters led to the conclusion that the structural genes arcA (for arginine deiminase), arcB (for catabolic ornithine carbamoyltransferase) and arcC (for carbamate kinase) are contiguous and transcribed in the same direction. Thus, the arcABC cluster appears to have the characteristics of an operon. In Escherichia coli the cloned arcABC genes were expressed at low, non-inducible levels; strong vector promoters enhanced arc expression up to 100-fold. This indicates that transcriptional initiation at the arc promoter(s) is poor in E. coli.