Two genes encode (1----3, 1----4)-beta-glucan 4-glucanohydrolase (EC 3.2.1.73) isoenzymes in barley. A gene for isoenzyme EI has been isolated from a barley genomic library and the nucleotide sequence of a 4643 bp fragment determined. The gene is located on barley chromosome 5 while the gene for (1----3, 1----4)-beta-glucanase isoenzyme EII is carried on chromosome 1. The isoenzyme EI gene contains a single 2514 bp intron that is inserted in codon 25 of a sequence encoding a signal peptide of 28 amino acids. The coding region of the mature enzyme is characterized by a high G+C content, which results from an extreme bias towards the use of these nucleotides in the wobble base position of codons. Determination of the nucleotide sequence of the gene has enabled the complete primary structure of the enzyme to be deduced: isoenzyme EI shows 92% positional identity with the primary sequence of (1----3, 1----4)-beta-glucanase isoenzyme EII at both the nucleotide and amino acid level. However, the nucleotide sequences of the two genes diverge markedly in their 3' untranslated regions. Expression sites of the two genes were defined by Northern analysis using oligonucleotide probes specific for these 3' untranslated regions and by amplifying specific cDNAs through the polymerase chain reaction. In the tissues examined, transcription of the isoenzyme EII gene is restricted to the aleurone layer of germinated grain. In contrast, the gene for isoenzyme EI is transcribed at relatively high levels in young leaves, but also in the scutellum and aleurone of germinated grain.
Expression sites of genes encoding (1→3,1→4)-β-glucan 4-glucanohydrolase (EC 3.2.1.73) have been mapped in germinated barley grains (Hordeum vulgare L.) by hybridization histochemistry. A(32)P-labelled cDNA (copy DNA) probe was hybridized to cryosections of intact barley grains to localize complementary mRNAs. No mRNA encoding (1→3,1→4)-β-glucanase is detected in ungerminated grain. Expression of (1→3,1→4)-β-glucanase genes is first detected in the scutellum after 1 d and is confined to the epithelial layer. At this stage, no expression is apparent in the aleurone. After 2 d, levels of (1→3,1→4)-β-glucanase mRNA decrease in the scutellar epithelium but increase in the aleurone. In the aleurone layer, induction of (1→3,1→4)-β-glucanase gene expression, as measured by mRNA accumulation, progresses from the proximal to distal end of the grain as a front moving away from, and parallel to, the face of the scutellum.
A simple and precise method suitable for the routine determination of starch and p-glucan in barley and malt is described. Perchloric acid (50 mM) was used to effect rapid (3 min) and exhaustive extraction of both glucans which were then measured directly from this single extract by specific enzymic hydrolysis of the individual glucans to glucose. The glucose was also measured enzymically. Little or no acid hydrolysis of starch or p-glucan was observed under the extraction conditions used; most or all of the free glucose could be attributed to hydrolysis of sucrose. Complete solubilisation of the gum and hemicellulosic components of p-glucan was achieved. Preincubation of the acid extracts with protease prior to amyloglucosidase digestion resulted in higher measure ments (approximately 4% w/w) of starch. The method was used to measure the levels of starch and p-glucan in five varieties of barley with contrasting malting quality, in micro-malts prepared from these samples and in commercial lager and ale malts.
1When cultures of Schizosaccharomyces pombe growing exponentially in a minimal medium were treatedlwith 50 mM-EDTA for 1 h, nucleic acid synthesis was inhibited but protein synthesis was not. On transfer to fresh medium, 50% of the cells divided synchronously. These phenomena may be explained on the basis of reduced availability of Mg2+ following chelation with EDTA. I N T R O D U C T I O NCell division in yeast may be synchronized by altering intracellular Mg2+ and Ca2+ concentrations using the ionophore A23 187 (Duffus & Paterson, 1974a;Penman & Duffus, 1975). We have postulated that a continuous fall in intracellular Mg2+ concentration through the cell cycle may play a crucial part in the volume-related regulation of cell division (Duffus & Paterson,"l974b). If this is so, any factor which alters the intracellular Mg2+ concentration to a constantIleve1 may be expected to cause division synchrony. Thus we decided to test the effect of the divalent cation chelating agent EDTA, which might be expected to lower the free Mg2+ concentration in both the growth medium and the cells. METHODSSchizosaccharomyces pombe, NCYC 132, ATCC 24751, was grown in Edinburgh Minimal Medium No. 2 (EMM2) (Mitchison, 1970) at 30 "C in an orbital incubator operating at 160 rev. min-l. Stock cultures were kept at 30 "C in 10 ml EMM2 and subcultured weekly. To induce synchronous division, analytical grade EDTA was added to exponentially growing cultures to a final concentration of 50 l l l~ and the cultures were incubated for a further 60 min. The cells were harvested by centrifuging at 30 "C at 2500 g for 10 min and washed rapidly, by resuspension with 50 ml distilled water at room temperature and further centrifugation, before being re-inoculated into fresh EMM2 at 30 "C. Control cultures, without EDTA, were treated identically.Cell mass increase was followed by measuring absorbance at 595 nm. Cell numbers were determined using a Thoma haemocytometer, applying the convention described by Mitchison (1970) that cells should not be taken to have divided until a notch could be detected on either side of the cell plate.DNA was measured in yeasts from 100 ml culture. The cells were harvested by centrifuging at 2500 g for 10 min, resuspended in 10 ml 10 % (v/v) perchloric acid and stored at -18 "C overnight. On thawing, the cells were pelleted by centrifuging as before, resuspended in 5 ml 10 % (v/v) perchloric acid and allowed to stand at 0 "C for 60 min before recentrifuging. The pellets were extracted twice with 5 ml ethanol (96 %, V/V) at 80 "C for 5 min and then nucleic acids were extracted in 2 m15 % (v/v) perchloric acid. (This volume might
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