Molecular cloning and analysis of cDNA sequences derived from poly A+ RNA from barley endosperm: identification of B hordein related clones.

A collection of over 130 cDNA clones has been constructed in the bacterial plasmids pPH207 and pBR322 using as template the poly A+ RNA from membrane-bound polysomes of barley endosperm (cv. Sundance). Fifty four B hordein cDNA clones have been identified by cross-hybridization analysis and in vitro translation of plasmid-selected mRNAs. Hybridization of 11 of the B hordein cDNA clones to Northern blots of size-fractionated RNA indicated that the B hordein mRNA is ca. 1300 nucleotides long. One cDNA clone, pHvE-c16, has been partially sequenced and shown by comparison with C-terminal and other peptide sequences to be related to B1 hordein polypeptides. The results obtained from the analysis of the B hordein cDNA clones support the idea that the Hor 2 locus, which specifies the B hordeins, is complex and codes for a family of related mRNA species.     

Molecular analysis of a mutation conferring the high-lysine phenotype on the grain of barley (Hordeum vulgare)

We have analyzed the molecular nature of the Riso 56 mutation that occurs in barley. This mutation results in a depression of hordein accumulation in the grain and consequently in a higher overall lysine content. In particular, the amount of B hordein, which is encoded by the complex locus Hor-2, is decreased by about 75% because of the absence of the major components. The synthesis of certain minor polypeptides, with properties similar to the major B hordeins, remains unaffected. Analysis of endosperm RNA, by in vitro translation and hybridization to various cloned cDNAs derived from hordein mRNA, shows that mRNA for the major B hordeins is not present in the endosperm. Hybridization of a B hordein cDNA clone to gel-fractionated restriction digests of mutant and wild-type DNA indicates that at least 85 kb of DNA has been deleted from the Hor-2 locus in the high-lysine mutant.     

Quantification of Hordeins by ELISA: The Correct Standard Makes a Magnitude of Difference

Background

Coeliacs require a life-long gluten-free diet supported by accurate measurement of gluten (hordein) in gluten-free food. The gluten-free food industry, with a value in excess of $6 billion in 2011, currently depends on two ELISA protocols calibrated against standards that may not be representative of the sample being assayed.

Aim

The factors affecting the accuracy of ELISA analysis of hordeins in beer were examined.

Results

A simple alcohol-dithiothreitol extraction protocol successfully extracts the majority of hordeins from barley flour and malt. Primary hordein standards were purified by FPLC. ELISA detected different classes of purified hordeins with vastly different sensitivity. The dissociation constant (Kd) for a given ELISA reaction with different hordeins varied by three orders of magnitude. The Kd of the same hordein determined by ELISA using different antibodies varied by up to two orders of magnitude. The choice of either ELISA kit or hordein standard may bias the results and confound interpretation.

Conclusions

Accurate determination of hordein requires that the hordein standard used to calibrate the ELISA reaction be identical in composition to the hordeins present in the test substance. In practice it is not feasible to isolate a representative hordein standard from each test food. We suggest that mass spectrometry is more reliable than ELISA, as ELISA enumerates only the concentration of particular amino-acid epitopes which may vary between different hordeins and may not be related to the absolute hordein concentration. MS quantification is undertaken using peptides that are specific and unique enabling the quantification of individual hordein isoforms.     

Differential Protein Accumulation during Barley Grain Development

Barley grains were analysed during development for the presenceof salt-soluble proteins, hordeins and glutelins. Characteristictemporal differences between the fractions were observed withhordeins being produced relatively late during maturation. Analysisof this fraction by gel electrophoresis revealed differentialaccumulation of its component polypeptides. The C hordeins madeup a relatively higher percentage of total hordein at the earlystages, decreasing from 20% of the total at 33% final dry weightto 15% at maturity. The relative amount of the lowest molecularweight B hordein band (Bl) increased throughout developmentfrom 30% of the total at 33% final dry weight to about 45% atmaturity. Electrophoresis of the salt-soluble fraction showedthat a group of low molecular weight polypeptides appeared atthe same stage of development as did the hordeins. There wereonly relatively minor changes in the polypeptide compositionof the glutelin fraction.     

Heritable somaclonal variation in wild barley (Hordeum spontaneum)

Summary Progenies of H. spontaneum plants regenerated from immature embryo derived calli were analysed for somaclonal variation on the following traits: (1) organization of the intergenic spacer of the rRNA genes; (2) B and C hordein pattern on SDS-PAGE; (3) genomic organization of the B and C hordein coding sequences; (4) mitochondrial DNA organization assayed by hybridization of Southern blots of total DNA with mitochondrial coding genes; (5) cytology. One out of twelve progeny plants was characterized as variant for two traits: (a) a loss of 1.8 and 2.5 kb Taq I intergenic rDNA spacer fragments and (b) a variant pattern of hordeins on 1-D SDS-PAGE. No numerical or structural chromosome variation was detected among the control plants therefore it is assumed that the variation was caused by the in vitro culture and transmitted, through sexual reproduction, to the analysed progeny.     

Cloning, characterization, and tissue distribution of messenger RNA species expressed at moderate and scarce frequencies within the lactating guinea pig mammary gland

In the lactating guinea pig mammary gland, the most abundant mRNA species encoding the major milk proteins, alpha-lactalbumin and caseins A, B, and C, have been extensively studied. Here we describe the isolation and characterization of cloned cDNA sequences representative of moderately abundant and scarce mammary gland mRNA species present at estimated concentrations of 1,400 (pgpO5), 540 (pgpKE6), 36 (pgpK1), and 2 (pgpJF4) copies per sequence per cell. RNA blotting showed these to represent mRNA species of 1,150, 1,900, 1,250, and 3,300 nucleotides in size, respectively. Hybrid selection cell-free synthesis showed that the mRNAs encoded proteins of Mr 33,000 (pgpO5), 58,000 (pgpKE6), and 36,000 (pgpK1). Studies on the tissue distribution of mammary gland mRNAs showed that the mRNA species of lower abundance, but not milk protein mRNAs, were expressed in other tissues but at concentrations differing from those in the mammary gland. None were expressed in all tissues, and so were not typical "housekeeping" proteins. We have used these cloned cDNA species to reinvestigate the apparent differential accumulation of moderately abundant poly(A)-containing mRNA species in polyadenylated and nonpolyadenylated cytoplasmic RNA populations of the mammary gland. Unlike previous observations, based on RNA excess hybridization using fractionated cDNA probes, the use of sequence-specific cloned cDNA probes showed that little intact mRNA was present in the nonpolyadenylated fraction. Thus previous observations were a reflection of the preferential accumulation of fragments of moderately abundant mRNA species, possibly a result of enhanced turnover. The significance of our results in terms of future investigations into factors which determine mRNA accumulation and tissue-specific expression is discussed.     

Ovarian cysteine proteinases in the teleost Fundulus heteroclitus: molecular cloning and gene expression during vitellogenesis and oocyte maturation

The cysteine proteinases cathepsins B and L are members of the multigene family of lysosomal proteases that have been implicated in the processing of yolk proteins (YPs) in teleost oocytes. However, the full identification of the type of cathepsins expressed in fish ovarian follicles and embryos, as well as their regulatory mechanisms and specific function(s), are not yet elucidated. In this study, cDNAs encoding cathepsins B, L, F, K, S, Z, C, and H have been isolated from the teleost Fundulus heteroclitus, and the analysis of their deduced amino acid sequences revealed highly similar structural features to vertebrate orthologs, and confirmed in this species the existence of cathepsin L-like, cathepsin B-like, and cathepsin F-like subfamilies of cysteine proteinases. While all identified cathepsins were expressed in ovarian follicles, the corresponding mRNAs showed different temporal expression patterns. Thus, similar mRNA levels of cathepsins L, F, S, B, C, and Z were found throughout the oocyte growth or vitellogenesis period, whereas those for cathepsin H and K appeared to decrease as vitellogenesis advanced. During oocyte maturation, a transient accumulation of cathepsins L, S, H, and F mRNAs, approximately a 3-, 1.5-, 1.6-, and 6-fold increase, respectively, was detected in ovarian follicles within the 20-25 hr after hormone stimulation, coincident with the maximum proteolysis of the oocyte major YPs. The specific temporal pattern of expression of these genes may indicate a potential role of cathepsin L-like and cathepsin F proteases in the YP processing events occurring during fish oocyte maturation and/or early embryogenesis.     

Hordein variation in the genus Hordeum as recognized by monoclonal antibodies.

The composition of the major storage protein, hordein, in wild barley species has been studied by using gel electrophoresis, Coomassie staining, and immunoblot assays. We have shown earlier that it is possible to obtain cross-reaction outside the cultivated barley, with monoclonal antibodies raised against hordeins from the barley cultivar Bomi. These antibodies have now been used to investigate the hordein composition in all species of the Hordeum genus. The results showed that polypeptides similar to the two major hordein groups of cultivated barley, the B- and C-hordeins, are produced in all wild Hordeum species, and that there are both similarities and differences between the two hordein groups. The similarities indicate a common evolutionary origin, while the distinction between B- and C-hordeins in the entire genus clearly shows that the divergence of their coding genes preceded the divergence of the Hordeum species. The presence of the same antigenic site in two different species indicates that they are evolutionarily related. Among the wild species, two rarely occurring sites were exclusively found in H. vulgare ssp. spontaneum and H. bulbosum, which confirms that they are the cultivated barley's closest relatives. Some of the antibodies also gave an extensive reaction pattern with H. murinum, which suggests a fairly close relationship to H. vulgare, though not as close as between H. vulgare and H. bulbosum.     

Measuring Hordein (Gluten) in Beer – A Comparison of ELISA and Mass Spectrometry

Background

Subjects suffering from coeliac disease, gluten allergy/intolerance must adopt a lifelong avoidance of gluten. Beer contains trace levels of hordeins (gluten) which are too high to be safely consumed by most coeliacs. Accurate measurement of trace hordeins by ELISA is problematic.

Methods

We have compared hordein levels in sixty beers, by sandwich ELISA, with the level determined using multiple reaction monitoring mass spectrometry (MRM-MS).

Results

Hordein levels measured by ELISA varied by four orders of magnitude, from zero (for known gluten-free beers) to 47,000 µg/mL (ppm; for a wheat-based beer). Half the commercial gluten-free beers were free of hordein by MS and ELISA. Two gluten-free and two low-gluten beers had zero ELISA readings, but contained significant hordein levels (p<0.05), or near average (60–140%) hordein levels, by MS, respectively. Six beers gave false negatives, with zero ELISA readings but near average hordein content by MS. Approximately 20% of commercial beers had ELISA readings less than 1 ppm, but a near average hordein content by MS. Several barley beers also contained undeclared wheat proteins.

Conclusions

ELISA results did not correlate with the relative content of hordein peptides determined by MS, with all barley based beers containing hordein. We suggest that mass spectrometry is more reliable than ELISA, as ELISA enumerates only the concentration of particular amino-acid epitopes; this may vary between different hordeins and may not be related to the absolute hordein concentration. MS quantification is undertaken using peptides that are specific and unique, enabling the quantification of individual hordein isoforms. This outlines the problem of relying solely on ELISA determination of gluten in beverages such as beer and highlights the need for the development of new sensitive and selective quantitative assay such as MS.     

Prediction of protein cleavage sites by the barley cysteine endoproteases EP-A and EP-B based on the kinetics of synthetic peptide hydrolysis

Hordeins, the natural substrates of barley (Hordeum vulgare) cysteine endoproteases (EPs), were isolated as protein bodies and degraded by purified EP-B from green barley malt. Cleavage specificity was determined by synthesizing internally quenched, fluorogenic tetrapeptide substrates of the general formula 2-aminobenzoyl-P(2)-P(1)-P(1)'-P(2)' 1-tyrosine(NO(2))-aspartate. The barley EPs preferred neutral amino acids with large aliphatic and nonpolar (leucine, valine, isoleucine, and methionine) or aromatic (phenylalanine, tyrosine, and tryptophan) side chains at P(2), and showed less specificity at P(1), although asparagine, aspartate, valine, and isoleucine were particularly unfavorable. Peptides with proline at P(1) or P(1)' were extremely poor substrates. Cleavage sites with EP-A and EP-B preferred substrate sequences are found in hordeins, their natural substrates. The substrate specificity of EP-B with synthetic peptides was used successfully to predict the cleavage sites in the C-terminal extension of barley beta-amylase. When all of the primary cleavage sites in C hordein, which occur mainly in the N- and C-terminal domains, were removed by site-directed mutagenesis, the resulting protein was degraded 112 times more slowly than wild-type C hordein. We suggest that removal of the C hordein terminal domains is necessary for unfolding of the beta-reverse turn helix of the central repeat domain, which then becomes more susceptible to proteolytic attack by EP-B.     

Triiodothyronine stimulates cystatin C production in bone cells

Thyroid hormones increase cystatin C levels in vivo. To study whether 3,3',5-triiodo-l-thyronine (T(3)) stimulates the production of cystatin C in vitro, we used a T(3)-responsive osteoblastic cell line (PyMS) which can be kept in serum-free culture. We compared the effects of T(3) on cystatin C mRNA expression (by Northern) and on protein release (by Western and ELISA) with those of dexamethasone (dex). Triiodothyronine increased cystatin C mRNA expression and cystatin C accumulation in culture media in a dose- and time-dependent manner, 1.5-fold at 1 nmol/l after 4d; dex (100 nmol/l) was more potent and increased cystatin C accumulation 3-fold after 4d. Triiodothyronine but not dex stimulated glucose uptake. Our in vitro findings explain in vivo observations. Triiodothyronine-induced increase in the production of cystatin C may be related to an increased cell metabolism and proteolysis control demand.