Sequences variation and classification of B-hordein genes in hull-less barley from the Qinghai-Tibet Plateau

The goal of this study is to understand the evolution relationship of the members of the B-hordein gene family in hull-less barley by analysis of their structure and to explore their utility in grain quality improvement. Six copies of the B-hordein gene (Hn1-Hn3, Hn7-Hn9) were cloned from six hull-less barley cultivars collected from Qinghai-Tibet Plateau and molecularly characterized. Comparison of their predicted polypeptide sequences with the published data suggested that they all share the same basic protein structures. In addition, we found that the C-terminal end sequences of all B-hordeins shared a similar feature. In the six clones and the other three published genes (Hn4, Hn5, and Hn6) from hull-less barley, Hn2 and Hn7 contained the identical C-terminal end sequence DIMPVDFWH. Hn3, Hn4, Hn5, Hn8 and Hn9 also shared the common sequence DIMPPDFWH, which was similar to that of a B-hordein reported previously. Both Hn1 and Hn6 exhibited differences in their C-terminal end sequences, and they clustered into different subgroups. The B-hordeins with identical C-terminal end sequences were clustered into the same subgroup, so we believe that B-hordein gene subfamilies possibly can be classified on the basis of the conserved C-terminal end sequences of predicted polypeptide. Phylogenetic analysis also indicated that there is a relatively weak identity between our predicted B-hordeins and those reported from H. chilense and H. brevisubulatum. All of our nine predicted B-hordeins were clustered together and other B-hordeins formed another cluster. The possible use of these genes in relation to barley quality is discussed. Published in Russian in Molekulyarnaya Biologiya, 2008, Vol. 42, No. 1, pp. 63–70. The text was submitted by the authors in English     

Cloning and Characterization of Four B-hordein Genes from Tibetan Hull-less Barley (Hordeum vulgare subsp. vulgare)

Four B-hordein genes, designated BH1-BH4, were cloned using PCR amplification from two hull-less barley cultivars, ZQ7239 and ZQ148, collected from Tibet. The results of sequencing indicated that BH1-BH4 contained complete open reading frames (ORFs). Comparison of their predicted polypeptide sequences with the published sequences suggested that they all share the same basic protein structure. Phylogenetic analysis indicated that the deduced amino-acid sequences of BH1-BH4 genes were more closely related to B-hordeins from cultivated barley (Hordeum vulgare L.) than to any other prolamins from wild barley and Aegilops tauschii. Comparison of the coding regions of BH1-BH4 genes showed that BH1 had a lower sequence identity to other previously published B-hordeins than the other three B-hordeins obtained in this study. BH1 was then cloned in a bacterial expression vector based on bacteriophage T7 RNA polymerase. The resulting plasmid produced a 28.15 kDa protein in Escherichia coli. The potential value of B-hordein genes in grain quality improvement of hull-less barley has been discussed.     

西藏青稞4个B组醇溶蛋白基因的克隆和特征

从两份西藏青稞材料中分离克隆出4个B组醇溶蛋白基因（BH1—BH4）,DNA测序结果表明：它们均包含完整的开放阅读框。推断的氨基酸序列与先前报道的大麦B组醇溶蛋白具有相同的蛋白质基本结构。系统分析表明：它们推断的氨基酸序列与栽培大麦中的B组醇溶蛋白具有较高的相关性,与野生大麦和山羊草属的醇溶谷蛋白相似性较低。并且,在4个基因BH1—BH4中,BH1与先前报道的B组醇溶蛋白基因有较低的序列相似性,因此我们对BH1基因进行了原核表达,含该基因的表达载体在大肠杆菌中表达出相对分子质量为28．15kDa并以包涵体形式存在的蛋白,进一步对其在青稞谷粒品质改良中的潜在价值进行了探讨。     

Structural and expressional analysis of the <Emphasis Type="Italic">B-hordein</Emphasis> genes in Tibetan hull-less barley

The B-hordein gene family was analyzed from two Tibetan hull-less barley cultivars Z09 and Z26 (Hordeum vulgare subsp. vulgare). Fourteen B-hordein genes, designated BZ09-2 to BZ09-5 (from Z09) and BZ26-1 to BZ26-10 (from Z26), were sequenced. Seven of them, similar to a previously reported BZ09-1 from Z09, were predicted to encode putative active proteins each with a signal peptide, a repetitive domain, and a C-terminal region; seven of them were predicted to be pseudogenes. The B-hordein gene family was analyzed using all known representatives of B-hordein sequences and two most similar LMW-GSs of Triticum aestivum. Alignment of these seven putative proteins with known B-hordeins and two most similar LMW-GSs of T. aestivum revealed that they shared a common motif. A large variation was observed between numbers of repeat units of predicted B-hordeins of Z26 and Z09. Phylogenetic analysis revealed that all BZ26 clones were clustered in a subgroup, and BZ09-1 formed another subgroup by itself in the putative eight active genes. In addition, six 5′-upstream regulatory sequences of the B-hordein genes were isolated from the two accessions by a single oligonucleotide nested PCR, and several different mutations were identified in the cis-acting element GLM and two distinctive sequences (Z09P-2 and Z26P-3). Phylogenetic analysis of 5′-upstream regulatory regions of the B-hordein genes showed that members from the same accession were clustered together except for two distinct members. Quantitative real time PCR analysis indicated distinct expression levels of B-hordein genes in four developing stages of developing grains in two accessions. These findings suggest B-hordein genes have intrinsic differences between accessions, and this knowledge will be useful for incorporating the B-hordeins protein in barley breeding programs.     

Short tandem repeats shared by B- and C-hordein cDNAs suggest a common evolutionary origin for two groups of cereal storage protein genes

We have identified cDNA clones coding for the major sulphur-rich and sulphur-poor groups of barley storage proteins (the B- and C-hordeins, respectively). Hybridization studies have revealed unexpected homologies between B- and C-hordein mRNAs. Using a deletion mutant (Risø 56), we have mapped some C-hordein-related sequences within, or closely associated with, B-hordein genes at the Hor 2 locus. Nucleotide sequencing has shown that the primary structure of B-hordein polypeptides can be divided into at least two domains: domain 1 (repetitive, proline-rich, sulphur-poor), which is homologous to C-hordein sequences, and domain 2 (non-repetitive, proline-poor, sulphur-rich), which makes up two-thirds of the polypeptide and is partially homologous to a 2S globulin storage protein found in dicotyledons. The coding sequences that are homologous in B- and C-hordein mRNAs have an asymmetric base composition (>80% C-A) and are largely composed of a degenerate tandem repeat based on a 24 nucleotide consensus that encodes Pro-Gln-Gln-Pro-Phe-Pro-Gln-Gln. We discuss the evolutionary implications of the domain structure of the B-hordeins and the unusual relationship between the two groups of barley storage proteins.     

Nucleotide sequence of a B1 hordein gene and the identification of possible upstream regulatory elements in endosperm storage protein genes from barley, wheat and maize

The B-hordeins are the major group of prolamin storage proteins in barley (Hordeum vulgare L.) and they are encoded by a small multigene family that is expressed specifically in the developing endosperm. We report the complete nucleotide sequence of a clone of one B-hordein gene (pBHR184). The cloned gene contains no introns and belongs to the B1 sub-family of B-hordein genes. Comparison of the 5'-flanking sequences of pBHR184 with those of related S-rich prolamin genes from wheat shows that several short sequences within 600 bp upstream of the translation initiation codon are strongly conserved. A sequence that is conserved at around -300 bp in the S-rich prolamins is also conserved at similar locations in genes encoding the two major classes of maize prolamin (the Z19 and Z21 zeins) and appears to be unique to prolamin genes. We discuss the possible role of this '-300 element' in the control of gene expression in the developing cereal endosperm.     

Prolamin variation and evolution in Triticeae as recognized by monoclonal antibodies.

The isopropanol-soluble seed storage proteins, prolamins, were studied in 18 different genera of the tribe Triticeae by gel electrophoresis, Coomassie staining, and immunoblot assays. The monoclonal antibodies were originally raised against cultivated barley (Hordeum vulgare L.) hordein, and their reactions had been tested earlier on wild Hordeum species. The study showed that all the investigated Triticeae species produce prolamins and that structural similarities can be found throughout the tribe. The presence of the same antigenic sites in all the species indicates that the polypeptides contain well-conserved regions. They also indicate that the prolamins of the Triticeae species have a common evolutionary origin. In all the investigated species an antigenic site that is common to the B- and C-hordeins of barley was detected. Some of the reacting polypeptides also contained a site that is only present in the B-hordeins. The B-hordein specific site was found in all genera except Agropyron, Hordelymus, and Secale. This shows that although there are similarities between individual polypeptides, the composition of the various prolamin groups may vary between different genera. In the polyploid Elymus species different banding patterns were observed depending on what basic genomes were represented. The results suggest a direct correlation between the presence of a fast migrating polypeptide containing the B-hordein specific site and the presence of the H genome.     

Localization of the B-hordein locus on barley chromosomes using fluorescence in situ hybridization

The B-hordein gene family consists of at least 13 genes that appear to be clustered in two regions on the barley chromosome 1H (5). Using fluorescence in situ hybridization techniques, we have localized the B-hordein locus (Hor2) to the distal end of the short arm of chromosome 1H (5), corresponding to the genetically determined position, 91% distal on the short arm. This result is based on appropriate barley lines (i.e. the two cultivars Igri and Betzes, the telotrisomic and ditelotetrasomic lines 1HS and the translocation line T21), on two experimental approaches and on a signal frequency of between 20% and 60% on metaphase chromosomes.     

The primary structure of a 4.0-kDa photosystem I polypeptide encoded by the chloroplast psaI gene

Partial amino acid sequences have been determined for a 4.0-kDa photosystem I polypeptide from barley. A comparison with the sequence of the chloroplast genome of Nicotiana tabacum and Marchantia polymorpha identified the polypeptide as chloroplast-encoded. We designate the corresponding gene psaI and the polypeptide PSI-I. The barley chloroplast psaI gene was sequenced. The gene encodes a polypeptide of 36 amino acid residues with a deduced molecular mass of 4008 Da. The 4.0-kDa polypeptide is N-terminally blocked with a formyl-methionine residue. Plasma desorption mass spectrometry established that the polypeptide is not post-translationally processed except for possible conversion of a methionine residue into methionine sulfone. The hydrophobic 4.0-kDa polypeptide is predicted to have one membrane-spanning alpha-helix and is homologous to transmembrane helix E of the D2 reaction center polypeptide of photosystem II.     

Sequence analysis and characterization of a maize gene encoding a high-sulfur zein protein of Mr 15,000

We have isolated a gene coding for a sulfur-rich zein protein from a maize genomic library. The nucleotide sequence of this gene predicts a protein composed of 180 amino acids, including a 20-amino acid signal peptide. As is true of other zeins, there are no intervening sequences in the gene. Comparison of the nucleotide sequence of this gene with that of a homologous cDNA clone revealed only a single difference resulting in a valine/alanine substitution. The Mr 15,000 zein contains no repetitive nucleotide sequences and shows no homology with genes encoding the Mr 22,000 and 19,000 zeins. Circular dichroism analysis of the Mr 15,000 zein protein revealed that it is composed primarily of beta and turn structures. This gene has a short region of nucleotide sequence homology to the cysteine-rich domain of the Mr 27,000 zein, as well as the cysteine-containing barley B-hordein.     

Myosin I heavy-chain genes of Acanthamoeba castellanii: cloning of a second gene and evidence for the existence of a third isoform

We have determined the complete sequence and structure of a second myosin I heavy-chain gene from Acanthamoeba castellanii. This gene, which we have named MIL, spans approx. 6kb, is split by 17 introns, encodes a 1147-aa polypeptide, and is transcribed in log-phase cells. The positions of six of the introns are conserved relative to a vertebrate muscle myosin gene. Similar to the previously characterized MIB heavy-chain gene, the deduced MIL heavy-chain aa sequence reveals a 125-kDa protein composed of a myosin globular head domain joined to a novel, approx. 50-kDa C-terminal domain that is rich in glycine, proline and alanine residues. There are differences, however, between MIL and MIB in the sequence organization of their unconventional C-terminal domains. We conclude from this and other data that Acanthamoeba express at least three myosin I heavy-chain isoforms: MIL, plus MIA and MIB, whose purifications have been published previously. Amoeba genomic DNA blots probed with a short, highly conserved sequence whose position is transposed between MIB and MIL indicate that the Acanthamoeba myosin I heavy-chain gene family may actually contain as many as six genes. Finally, we compared the myosin I sequences with those of two related proteins, Drosophila NinaC and the bovine myosin I-like protein, and found that a portion of the unconventional C-terminal domains of the amoeba myosins I and the bovine protein appear to be related.     

Sortase independent and dependent systems for acquisition of haem and haemoglobin in Listeria monocytogenes

We studied three Fur-regulated systems of Listeria monocytogenes: the srtB region, that encodes sortase-anchored proteins and a putative ABC transporter, and the fhu and hup operons, that produce putative ABC transporters for ferric hydroxamates and haemin (Hn)/haemoglobin (Hb) respectively. Deletion of lmo2185 in the srtB region reduced listerial [(59) Fe]-Hn transport, and purified Lmo2185 bound [(59) Fe]-Hn (K(D) = 12 nM), leading to its designation as a Hn/Hb binding protein (hbp2). Purified Hbp2 also acted as a haemophore, capturing and supplying Hn from the environment. Nevertheless, Hbp2 only functioned in [(59) Fe]-Hn transport at external concentrations less than 50 nM: at higher Hn levels its uptake occurred with equivalent affinity and rate without Hbp2. Similarly, deletion of sortase A had no effect on ferric siderophore or Hn/Hb transport at any concentration, and the srtA-independence of listerial Hn/Hb uptake distinguished it from comparable systems of Staphylococcus aureus. In the cytoplasmic membrane, the Hup transporter was specific for Hn: its lipoprotein (HupD) only showed high affinity for the iron porphyrin (K(D) = 26 nM). Conversely, the FhuD lipoprotein encoded by the fhu operon had broad specificity: it bound both ferric siderophores and Hn, with the highest affinity for ferrioxamine B (K(D) = 123 nM). Deletions of Hup permease components hupD, hupG or hupDGC reduced Hn/Hb uptake, and complementation of ΔhupC and ΔhupG by chromosomal integration of hupC(+) and hupG(+) alleles on pPL2 restored growth promotion by Hn/Hb. However, ΔhupDGC did not completely eliminate [(59) Fe]-Hn transport, implying the existence of another cytoplasmic membrane Hn transporter. The overall K(M) of Hn uptake by wild-type strain EGD-e was 1 nM, and it occurred at similar rates (V(max) = 23 pmol 10(9) cells(-1) min(-1)) to those of ferric siderophore transporters. In the ΔhupDGC strain uptake occurred at a threefold lower rate (V(max) = 7 pmol 10(9) cells(-1) min(-1)). The results show that at low (< 50 nM) levels of Hn, SrtB-dependent peptidoglycan-anchored proteins (e.g. Hbp2) bind the porphyrin, and HupDGC or another transporter completes its uptake into the cytoplasm. However, at higher concentrations Hn uptake is SrtB-independent: peptidoglycan-anchored binding proteins are dispensable because HupDGC directly absorbs and internalizes Hn. Finally, ΔhupDGC increased the LD(50) of L. monocytogenes 100-fold in the mouse infection model, reiterating the importance of this system in listerial virulence.     

Molecular analysis of an outer membrane protein, MopB, of Methylococcus capsulatus (Bath) and structural comparisons with proteins of the OmpA family

The gene encoding a major outer membrane protein (MopB) of the methanotroph Methylococcus capsulatus (Bath) was cloned and sequenced. The cloned DNA contained an open reading frame of 1044 bp coding for a 348-amino-acid polypeptide with a 21-amino-acid leader peptide. Comparative sequence analysis of the predicted amino acid sequence revealed that the C-terminal part of MopB possessed sequences that are conserved in the OmpA family of proteins. The N-terminal half of the protein had no significant sequence similarity to other proteins in the databases, but the predicted secondary structure showed stretches of amphipathic beta-strands typical of transmembrane segments of outer membrane proteins. A region with four cysteines similar to the cysteine-encompassing region of the OprF of Pseudomonas aeruginosa was found toward the C-terminal part of MopB. Results from whole-cell labeling with the fluorescent thiol-reacting reagent 5-iodoacetamidofluorescein indicated a surface-exposed location for these cysteines. A probe consisting of the 3'-end of the mopB gene hybridized to the type I methanotroph Methylomonas methanica S in Southern blots containing DNA from nine methanotrophic strains representing six different genera.     

The low-molecular-weight glutenin subunit proteins of primitive wheats. III. The genes from D-genome species

 The isolation and characterisation by DNA sequencing of two different low molecular weight glutenin subunit (LMW-GS) genes from a genomic library derived from Triticum tauschii is described. These genes are similar (more than 90% similarity) but not identical to previously published LMW-GS gene sequences from cultivated wheats. A comparison of nucleotide sequence of the coding regions revealed the presence of insertions and deletions preferentially located in the region encoding the domains in the LMW-GS proteins rich in proline and glutamine and the middle part of the C-domain. The signal sequences, the amino-terminus and the remaining parts of the C-domain were conserved between all the LMW-GSs compared. The differences detected between the deduced amino-acid sequences in these three regions are only due to single nucleotide substitutions. The most important characteristic of all compared LMW-GS genes is the conservation of eight cysteine residues that could be involved in potential secondary or tertiary structure and disulphide-bond interactions. Comparisons between the 5′ and 3′ non-coding sequences of one of the isolated clones (LMW-16/10) with those of different prolamin genes from wheat, barley and rye led to the distinction of five different gene families, and confirmed the evolutionary relationships determined previously for these genes mainly on the basis of the coding region. In particular, the LMW-GS sequences are more closely related to the B-hordein sequences than to any other prolamin genes from wheat, barley and rye. Formal proof that the isolated genes coded for LMW-GSs, as defined by gel electrophoresis, was obtained by moving one of these genes (LMW-16/10) into a bacterial expression vector based on bacteriophage T7 RNA polymerase. The resulting plasmid directed the synthesis of large amounts of the mature form of the subunit in Escherichia coli. This protein exhibited solubility characteristics identical to those of the LMW-GSs and cross-reacted with antibodies reactive with these proteins. Received: 24 November 1997 / Accepted: 18 August 1998     

Distribution of the ermG gene among bacterial isolates from porcine intestinal contents

The ermG gene was first found in the soil bacterium Bacillus sphaericus. More recently, it was found in several human intestinal Bacteroides species. We report here the first finding of ermG genes in gram-positive bacteria isolated from porcine feces and from under-barn manure pits used to store porcine wastes. The porcine ermG sequences were identical to the sequence of the B. sphaericus ermG gene except that six of the seven ermG-containing strains contained an insertion sequence element insertion in the C-terminal end of the gene. The porcine ermG genes were found in three different gram-positive genera, an indication that it is possible that the gene is being spread by horizontal gene transfer. A segment of a Bacteroides conjugative transposon that carries an ermG gene cross-hybridized with DNA from six of the seven porcine isolates, but the restriction patterns in the porcine strains were different from that of the Bacteroides conjugative transposon.     

Identification and nucleotide sequence of the Acinetobacter calcoaceticus encoded trpE gene

Summary The trpE gene from Acinetobacter calcoaceticus encoding the anthranilate synthase component I was cloned, identified by deletion analysis and sequenced. It encodes a predicted polypeptide of 497 amino acids with a calculated molecular weight of 55323. Its primary structure shows 49% identical amino acids with the enzyme from Clostridium thermocellum, 45% with that of Thermus thermophilus and only 35% with that of Escherichia coli. The codon usage of the trpE genes encoding the most homologous enzymes differs greatly indicating selection for amino acid maintainance. The homologies are clustered in the C-terminal 200 amino acids of the sequences indicating that this part is important for enzymic activity.     

Cloning and molecular characterization of B-hordeins from Hordeum chilense (Roem. et Schult.)

One of the main limitations of cereal breeding is the lack of genetic variability within cultivated crops. Hordeum chilense is a wild relative of Hordeum vulgare, which has been successfully used in the synthesis of amphiploids by crossing with Triticum spp. Among the agronomic traits of these new amphiploids, the allelic variation in the endosperm storage proteins and their influence on breadmaking and malting quality are of special interest. B-hordeins are sulfur rich prolamins, which account for 70–80% of the total hordein fraction in barley. In this work, rapid amplification of cDNA ends by PCR (RACE-PCR) has been used for the cloning of the full-length open reading frame (ORF) of six sequences of B3-hordeins from two lines of H. chilense. Two consensus sequences of 813 and 822 bp for the H1 and H7 lines, respectively, were determined by alignment of all the sequences generated. Between both lines, differences involving single base changes, which could correspond to single nucleotide polymorphisms (SNP), insertions and deletions were observed. Of these differences, only six out of the 13 within the ORF caused a change of amino acid. Two insertions/deletions of 9 and 12 bp were also observed between both lines. The derived amino acid sequences showed a similar structure to the B-hordeins from cultivated barley and other prolamins. The repetitive region is based on the repetition of the motif PQQPFPQQ. The copy number of the B3-hordeins was estimated as a minimum of nine and five copies for the H1 and H7 lines, respectively. The expression profile of the B-hordeins through the developing endosperm is also described in this work. This study of the storage proteins of H. chilense is a useful contribution to the knowledge of the genetic diversity available in wild relatives of cultivated barley. In addition, the origin of the different prolamins can be better understood with an in-depth knowledge of its wild equivalent.     

Molecular Characterization of a Distinct Begomovirus and its Associated Satellite DNA Molecule Infecting Sida acuta in China*

Three viral isolates Hn8, Hn40 and Hn41 were obtained from Sida acuta showing yellow mosaic symptom in the Hainan province, China. Comparison of partial DNA‐A sequences amplified with degenerate primers confirmed the existence of single type of Begomovirus. The complete nucleotide sequence of the DNA‐A‐like molecule of Hn8 was determined to be 2749 nucleotides, having a typical genetic organization of a Begomovirus. Hn8 DNA‐A had the highest sequence identity (78%) with that of Ageratum yellow vein China virus‐[G13] ( AJ558120 ), and had less sequence identity with other begomoviruses. Based on the above molecular data, Hn8 was thus considered as a new Begomovirus species, for which the name Sida yellow mosaic China virus (SiYMCNV) is proposed. Satellite DNA‐β molecules (Hn8‐β, Hn40‐β and Hn41‐β) were found to be associated with Hn8, Hn40 and Hn41 and their complete nucleotide sequences were determined. Sequence analysis showed that Hn8‐β, Hn40‐β and Hn41‐β shared more than 84% nucleotide sequence identity, and they were different from other characterized DNA‐β, sharing the highest nucleotide sequence identity (47.8%) with DNA‐β of Ageratum yellow vein virus.