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The influence of the malting barley genotype on the apparent attenuation limit (AAL) was investigated. The AAL level correlated closely with the maltose concentration in the wort but was not affected by other fermentable sugars or by the total carbohydrate content. The chemical composition, modification, amylolytic enzyme activities and several starch properties of selected malts were studied in detail. Variations in the maltose concentration could almost solely be traced back to genotype-dependent disparities of ␤-amylase thermostability. These differences are due to interallelic polymorphisms of the ␤-amylase gene and are easily detected by PCR. Hence, PCR primers offer remarkable prospects for breeding barley on the basis of a marker-assisted selection (MAS). Key words: Apparent attenuation limit, ␤-amylase, ␤-amylase thermostability, barley breeding, carbohydrates, fermentability, maltose. -2863(9'8-32In recent decades, malting barley breeding work has led to a considerable improvement in extract yield and modification. However, the extract yields currently attainable appear to be limited by the structure and chemical composition of the grain. Further attempts to enhance the extract may deteriorate barley quality, for example by an increased susceptibility to fungal attack caused by split husks. Efforts to improve malt quality are therefore intended to focus on increasing quality features of extract and thus its fermentability. The high heredity of the apparent attenuation limit (AAL) provides a good precondition for successful breeding work. However, the genetic basis of different AALs must first be unravelled.Expressing the percentage decline of 'extract' after complete fermentation, the AAL is intended to give information about the yield of fermentable sugars in the wort. It can be influenced by several factors as follows.A lack of extract and/or fermentable sugars in the wort can be due to a poor malt modification. If cell wall components and storage proteins are not sufficiently hydrolysed throughout the endosperm during malting, starch kernels will remain well embedded within the protein matrix and the interior of endosperm cell walls 23 . In this state, the granules gelatinise incompletely during mashing 23 and, hence, are less accessible for amylolytic enzymes 20 . A clear relationship between malt modification and the AAL could undoubtedly be confirmed both on a laboratory scale 11 and in brewing practice 12 . Differences in the extent of starch hydrolysis during mashing can also be affected by the starch itself. It has been ascertained that higher gelatinisation temperatures lead to a lower concentration of fermentable sugars in the wort 27 . This is obviously due to the ␤-amylase, as complete starch gelatinisation occurs at temperatures, where thermal inactivation of that enzyme has already occurred 27 . The ␤-amylase releases maltose from the non-reducing ends of ␣-1,4-linked poly-and oligoglucans 31 and is therefore considered essential for starch degradation during mashing. Both the characteris...

Stable genetic transformation of barley is routinely carried out by co-cultivation of immature zygotic embryos with Agrobacterium tumefaciens carrying binary vectors (1, 2). The target gene and the selection marker are located between the left and right 25-bp direct repeats of the T-DNA borders either within a single or in two adjacent tandemly arranged cassettes (3). The transfer of the T-DNA is polar; an intact right border is required for the transfer, whereas mutations in the left border have little effect on transfer efficiency (4). A single-stranded copy of the lower T-DNA strand is generated from endonucleolytic cleavage sites between the third and fourth bases of the border repeats. The strand is covered with the single-stranded DNA binding protein VirE2, covalently bound to the VirD2 protein at the 5Ј end and exported from the bacterium (4, 5). The VirE1 protein assists in secretion of virE2, which appears to form a voltage gated channel through the plasma membrane of the plant cell (6). The DNA is transported through the channel and guided by several proteins into the cell nucleus. The single-strand T-DNA then probably invades the DNA of the plant chromosome and is integrated by illegitimate recombination followed by secondstrand synthesis. Alternatively a double-stranded form might be generated first and then integrated into the chromosome (5).Details of integration have been primarily investigated in tobacco and Arabidopsis (7-10). Small deletions, base substitutions, duplicated border, and genomic sequences are found around the T-DNA͞plant DNA junctions. Co-integration into a chromosome locus after extrachromosomal second strand synthesis and ligation of the different T-DNA molecules, followed by docking and insertion via double-stranded breaks and repair synthesis is supported by analysis of co-integration of two or more T-DNAs in the same locus (11). All ten possible combinations-i.e., inverted as well as tandem arrangementsoccurred with regard to the left and right borders. The junction regions of direct repeats of T-DNA were investigated by selecting these with a vector containing a promoterless kanamycin phosphotransferase gene near the right border and a 35S CaMV promoter near the left border (12). Transformants from two T-DNA strands linking the promoter with the gene were selected on media containing kanamycin and revealed integrated T-DNA strands with a precise junction as well as integrations with 8 to 293 bp of filler DNA. Binary vectors carrying a neomycin phosphotransferase gene driven by a nopalinsynthase promoter within the T-DNA and a ␤-glucuronidase (gus) reporter gene under the control of a mannopine synthase promoter outside the left or right border resulted in 75% of transgenic tobacco plants containing the uidA (gus) gene unlinked or linked to the left or right border of the integrated T-DNA (13). Such incorporation of vector backbone sequences can be avoided by cotransformation of the target gene with the virD1, virD2, and virE2 virulence genes within the left and right border ...

Mature seeds of Cuphea lanceolata contain 83% capric acid in storage triacylglycerols (Graham, 1989). However, the regulation of the biosynthesis of medium-chain fatty acids in the genus Cuphea is not known. ACP plays an essential role as a cofactor in the plastid-located machinery of plant fatty acid biosynthesis. The growing acyl chain is covalently bound via a thioester to the ACP's 4'-phosphopantetheine group. Recently, protein sequences for two ACP isoforms of C. lanceolata have been reported (Kopka et al., 1993). We performed reverse transcriptase-PCR on C. lanceolata poly(A)+ RNA using degenerate oligonucleotides as primer pairs designed from these sequences. The resulting amplification product was used to screen a C. lanceolata embryo-specific XZAP-cDNA library. Here we present three complete cDNA clones encoding ACP (Table I).The probable full-size cDNAs cover, respectively, 810 bp (Ac1l;Cl-lu), 733 bp (Ac1l;Cl-lb), and 688 bp (Acl2;Cl-lc). The clones are very similar to each other; a comparison of the deduced amino acid sequences of the mature proteins revealed a homology of greater than 96%. The mature proteins corresponding to Acl2;CI-Za and AcZl;Cl-Zb are composed of 84 amino acids, whereas Acl2;Cl-lc is 83 amino acids long. In addition, each protein contains a Ser-rich transit peptide of 56 (Ac1l;Cl-lu), 53 (Acl2;CZ-lb), or 60 (AcZl;Cl-lc) amino acids, respectively. We predict that a11 clones contain between 30 and 49 nucleotides of 5' untranslated DNA, with the first ATG most likely representing the translational start site in a11 clones described. The predicted amino acid sequences based on the cDNAs a11 show strong homology to the protein sequence ACPl of Kopka et al. (1993); however, none is identical. The predicted stop codon is followed by 3' untranslated sequences of 319, 249, and 212 nucleotides,

Increasing contamination of environmental waters with pharmaceuticals represents an emerging threat for the drinking water quality and safety. In this regard, fast and reliable analytical methods are required to allow quick countermeasures in case of contamination. Here, we report the development of a magnetic bead-based immunoassay (MBBA) for the fast and cost-effective determination of the analgesic diclofenac (DCF) in water samples, based on diclofenac-coupled magnetic beads and a robust monoclonal anti-DCF antibody. A novel synthetic strategy for preparation of the beads resulted in an assay that enabled for the determination of diclofenac with a significantly lower limit of detection (400 ng/L) than the respective enzyme-linked immunosorbent assay (ELISA). With shorter incubation times and only one manual washing step required, the assay demands for remarkably shorter time to result (< 45 min) and less equipment than ELISA. Evaluation of assay precision and accuracy with a series of spiked water samples yielded results with low to moderate intra- and inter-assay variations and in good agreement with LC–MS/MS reference analysis. The assay principle can be transferred to other, e.g., microfluidic, formats, as well as applied to other analytes and may replace ELISA as the standard immunochemical method. Graphical abstract