Biomedical subjects

Andris Kleinhofs

Publications and source records attributed to Andris Kleinhofs.

13 recordsLinked to original sources

PubMed2006

A high-density consensus map of barley linking DArT markers to SSR, RFLP and STS loci and agricultural traits.

BACKGROUND: Molecular marker technologies are undergoing a transition from largely serial assays measuring DNA fragment sizes to hybridization-based technologies with high multiplexing levels. Diversity Arrays Technology (DArT) is a hybridization-based technology that is increasingly being adopted by barley researchers. There is a need to integrate the information generated by DArT with previous data produced with gel-based marker technologies. The goal of this study was to build a high-density consensus linkage map from the combined datasets of ten populations, most of which were simultaneously typed with DArT and Simple Sequence Repeat (SSR), Restriction Enzyme Fragment Polymorphism (RFLP) and/or Sequence Tagged Site (STS) markers. RESULTS: The consensus map, built using a combination of JoinMap 3.0 software and several purpose-built perl scripts, comprised 2,935 loci (2,085 DArT, 850 other loci) and spanned 1,161 cM. It contained a total of 1,629 'bins' (unique loci), with an average inter-bin distance of 0.7 +/- 1.0 cM (median = 0.3 cM). More than 98% of the map could be covered with a single DArT assay. The arrangement of loci was very similar to, and almost as optimal as, the arrangement of loci in component maps built for individual populations. The locus order of a synthetic map derived from merging the component maps without considering the segregation data was only slightly inferior. The distribution of loci along chromosomes indicated centromeric suppression of recombination in all chromosomes except 5H. DArT markers appeared to have a moderate tendency toward hypomethylated, gene-rich regions in distal chromosome areas. On the average, 14 +/- 9 DArT loci were identified within 5 cM on either side of SSR, RFLP or STS loci previously identified as linked to agricultural traits. CONCLUSION: Our barley consensus map provides a framework for transferring genetic information between different marker systems and for deploying DArT markers in molecular breeding schemes. The study also highlights the need for improved software for building consensus maps from high-density segregation data of multiple populations.

Chromosome Mapping↗

PubMed2006

Subcellular localization and functions of the barley stem rust resistance receptor-like serine/threonine-specific protein kinase Rpg1.

The Rpg1 gene confers resistance to many pathotypes of the stem rust fungus Puccinia graminis f. sp. tritici and has protected barley from serious disease losses for over 60 years. Rpg1 encodes a constitutively expressed protein with two tandem kinase domains. Fractionation by differential centrifugation and aqueous two-phase separation of the microsome proteins located Rpg1 mainly in the cytosol but also in the plasma membrane and intracellular membranes. Recombinant Rpg1 autophosphorylates in vitro intramolecularly only serine and threonine amino acids with a preference for Mn(2+) cations and a K(m) of 0.15 and a V(max) of 0.47 nmol.min(-1).mg(-1) protein. The inability of wild-type Rpg1 to transphosphorylate a recombinant Rpg1 inactivated by site-directed mutation confirmed that Rpg1 autophosphorylation proceeds exclusively via an intramolecular mechanism. Site-directed mutagenesis of the two adjacent lysine residues in the ATP anchor of the two-kinase domains established that the first of the two tandem kinase domains is nonfunctional and that lysine 461 of the second domain is the catalytically active residue. Transgenic barley, expressing Rpg1 mutated in either the kinase 1 or 2 domains, were fully susceptible to P. graminis f. sp. tritici revealing requirement of both kinase domains for resistance. In planta-expressed Rpg1 mutant protein confirmed that mutation in domain 2, but not 1, rendered the protein incapable of autophosphorylation.

Amino Acid Sequence↗

PubMed2006

An atlas of gene expression from seed to seed through barley development.

Assaying relative and absolute levels of gene expression in a diverse series of tissues is a central step in the process of characterizing gene function and a necessary component of almost all publications describing individual genes or gene family members. However, throughout the literature, such studies lack consistency in genotype, tissues analyzed, and growth conditions applied, and, as a result, the body of information that is currently assembled is fragmented and difficult to compare between different studies. The development of a comprehensive platform for assaying gene expression that is available to the entire research community provides a major opportunity to assess whole biological systems in a single experiment. It also integrates detailed knowledge and information on individual genes into a unified framework that provides both context and resource to explore their contributions in a broader biological system. We have established a data set that describes the expression of 21,439 barley genes in 15 tissues sampled throughout the development of the barley cv. Morex grown under highly controlled conditions. Rather than attempting to address a specific biological question, our experiment was designed to provide a reference gene expression data set for barley researchers; a gene expression atlas and a comparative data set for those investigating genes or regulatory networks in other plant species. In this paper we describe the tissues sampled and their transcriptomes, and provide summary information on genes that are either specifically expressed in certain tissues or show correlated expression patterns across all 15 tissue samples. Using specific examples and an online tutorial, we describe how the data set can be interrogated for patterns and levels of barley gene expression and how the resulting information can be used to generate and/or test specific biological hypotheses.

Databases, Genetic↗

PubMed2005

Barley necrotic locus nec1 encodes the cyclic nucleotide-gated ion channel 4 homologous to the Arabidopsis HLM1.

Barley homolog of the Arabidopsis necrotic (disease lesion mimic) mutant HLM1 that encodes the cyclic nucleotide-gated ion channel 4 was cloned. Barley gene was mapped genetically to the known necrotic locus nec1 and subsequent sequence analysis identified mutations in five available nec1 alleles confirming barley homolog of Arabidopsis HLM1 as the NEC1 gene. Two fast neutron (FN) induced mutants had extensive deletions in the gene, while two previously described nec1 alleles had either a STOP codon in exon 1 or a MITE insertion in intron 2 which caused alternative splicing, frame shift and production of a predicted non-functional protein. The MITE insertion was consistent with the reported spontaneous origin of the nec1 Parkland allele. The third FN mutant had a point mutation in the coding sequence which resulted in an amino acid change in the conserved predicted cyclic nucleotide-gated ion channel pore region. The expression of two pathogenesis-related genes, HvPR-1a and beta-1,3-glucanase, was elevated in two FN necrotic lines. Ten other members of the barley cyclic nucleotide-gated ion channel gene family were identified and their position on barley linkage map is reported.

Alleles↗

PubMed2004

Quantitative relationship between synonymous codon usage bias and GC composition across unicellular genomes.

BACKGROUND: Codon usage bias has been widely reported to correlate with GC composition. However, the quantitative relationship between codon usage bias and GC composition across species has not been reported. RESULTS: Based on an informatics method (SCUO) we developed previously using Shannon informational theory and maximum entropy theory, we investigated the quantitative relationship between codon usage bias and GC composition. The regression based on 70 bacterial and 16 archaeal genomes showed that in bacteria, SCUO = -2.06 * GC3 + 2.05*(GC3)2 + 0.65, r = 0.91, and that in archaea, SCUO = -1.79 * GC3 + 1.85*(GC3)2 + 0.56, r = 0.89. We developed an analytical model to quantify synonymous codon usage bias by GC compositions based on SCUO. The parameters within this model were inferred by inspecting the relationship between codon usage bias and GC composition across 70 bacterial and 16 archaeal genomes. We further simplified this relationship using only GC3. This simple model was supported by computational simulation. CONCLUSIONS: The synonymous codon usage bias could be simply expressed as 1+ (p/2)log2(p/2) + ((1-p)/2)log2((l-p)/2), where p = GC3. The software we developed for measuring SCUO (codonO) is available at http://digbio.missouri.edu/~wanx/cu/codonO.

Base Composition↗

PubMed2004

Diversity Arrays Technology (DArT) for whole-genome profiling of barley.

Diversity Arrays Technology (DArT) can detect and type DNA variation at several hundred genomic loci in parallel without relying on sequence information. Here we show that it can be effectively applied to genetic mapping and diversity analyses of barley, a species with a 5,000-Mbp genome. We tested several complexity reduction methods and selected two that generated the most polymorphic genomic representations. Arrays containing individual fragments from these representations generated DArT fingerprints with a genotype call rate of 98.0% and a scoring reproducibility of at least 99.8%. The fingerprints grouped barley lines according to known genetic relationships. To validate the Mendelian behavior of DArT markers, we constructed a genetic map for a cross between cultivars Steptoe and Morex. Nearly all polymorphic array features could be incorporated into one of seven linkage groups (98.8%). The resulting map comprised approximately 385 unique DArT markers and spanned 1,137 centimorgans. A comparison with the restriction fragment length polymorphism-based framework map indicated that the quality of the DArT map was equivalent, if not superior, to that of the framework map. These results highlight the potential of DArT as a generic technique for genome profiling in the context of molecular breeding and genomics.

Chromosome Mapping↗

PubMed2004

A new resource for cereal genomics: 22K barley GeneChip comes of age.

In recent years, access to complete genomic sequences, coupled with rapidly accumulating data related to RNA and protein expression patterns, has made it possible to determine comprehensively how genes contribute to complex phenotypes. However, for major crop plants, publicly available, standard platforms for parallel expression analysis have been limited. We report the conception and design of the new publicly available, 22K Barley1 GeneChip probe array, a model for plants without a fully sequenced genome. Array content was derived from worldwide contribution of 350,000 high-quality ESTs from 84 cDNA libraries, in addition to 1,145 barley (Hordeum vulgare) gene sequences from the National Center for Biotechnology Information nonredundant database. Conserved sequences expressed in seedlings of wheat (Triticum aestivum), oat (Avena strigosa), rice (Oryza sativa), sorghum (Sorghum bicolor), and maize (Zea mays) were identified that will be valuable in the design of arrays across grasses. To enhance the usability of the data, BarleyBase, a MIAME-compliant, MySQL relational database, serves as a public repository for raw and normalized expression data from the Barley1 GeneChip probe array. Interconnecting links with PlantGDB and Gramene allow BarleyBase users to perform gene predictions using the 21,439 non-redundant Barley1 exemplar sequences or cross-species comparison at the genome level, respectively. We expect that this first generation array will accelerate hypothesis generation and gene discovery in disease defense pathways, responses to abiotic stresses, development, and evolutionary diversity in monocot plants.

Edible Grain↗

PubMed2003

Chalcone isomerase gene from rice (Oryza sativa) and barley (Hordeum vulgare): physical, genetic and mutation mapping.

The barley and rice chalcone flavonone isomerase (Cfi) genes were isolated and identified by homology to the maize Cfi gene. Structure analysis indicated high similarity except that the barley gene lacked intron 3. The maize Cfi gene has been mapped to three loci, but only a single locus was detected in barley and rice. This explains the lack of observed mutants in maize while a single locus anthocyanin-less 30 (ant30), with four alleles ant30-245, ant30-310, ant30-272 and ant30-287 has been described in barley. Based on biochemical analysis it has been suggested that these mutants are in the Cfi gene resulting in absence of anthocyanin. In order to provide molecular evidence for or against this hypothesis we sequenced the four ant30 alleles and compared them to their respective wild-type alleles. The three sodium azide induced mutants ant30-245, ant30-272 and ant30-287 showed single base changes resulting in two non-sense and one mis-sense mutations affecting the protein function. The 1-nitroso-5,6-dihydrouracil induced mutant ant30-310 had one base substitution and a 25 bp deletion. These observations are in accordance with the conclusion that the ant30 phenotype is caused by mutations in the Cfi gene. The nature of the mutants induced is in line with the proposed mechanism of action for the mutagens used.

Chromosome Mapping↗

PubMed2002

Genetically engineered stem rust resistance in barley using the Rpg1 gene.

The stem-rust-susceptible barley cv. Golden Promise was transformed by Agrobacterium-mediated transformation of immature zygotic embryos with the Rpg1 genomic clone of cv. Morex containing a 520-bp 5' promoter region, 4,919-bp gene region, and 547-bp 3' nontranscribed sequence. Representatives of 42 transgenic barley lines obtained were characterized for their seedling infection response to pathotype Pgt-MCC of the stem rust fungus Puccinia graminis f. sp. tritici. Golden Promise was converted from a highly susceptible cultivar into a highly resistant one by transformation with the dominant Rpg1 gene. A single copy of the gene was sufficient to confer resistance against stem rust, and progenies from several transformants segregated in a 3:1 ratio for resistancesusceptibility as expected for Mendelian inheritance. These results unequivocally demonstrate that the DNA segment isolated by map-based cloning is the functional Rpg1 gene for stem rust, resistance. One of the remarkable aspects about the transformants is that they exhibit a higher level of resistance than the original sources of Rpg1 (cvs. Chevron and Peatland). In most cases, the Golden Promise transformants exhibited a highly resistant reaction where no visible sign of infection was evident. Hypersensitive necrotic "fleck" reactions were also observed, but less frequently. With both infection types, pathogen sporulation was prevented. Southern blot and RT-PCR analysis revealed that neither Rpg1 gene copy number nor expression levels could account for the increased resistance observed in Golden Promise transformants. Nevertheless, this research demonstrates that stem-rust-susceptible barley can be made resistant by transformation with the cloned Rpg1 gene.

Blotting, Southern↗

PubMed2002

Genomic sequencing reveals gene content, genomic organization, and recombination relationships in barley.

Barley (Hordeum vulgare L.) is one of the most important large-genome cereals with extensive genetic resources available in the public sector. Studies of genome organization in barley have been limited primarily to genetic markers and sparse sequence data. Here we report sequence analysis of 417.5 kb DNA from four BAC clones from different genomic locations. Sequences were analyzed with respect to gene content, the arrangement of repetitive sequences and the relationship of gene density to recombination frequencies. Gene densities ranged from 1 gene per 12 kb to 1 gene per 103 kb with an average of 1 gene per 21 kb. In general, genes were organized into islands separated by large blocks of nested retrotransposons. Single genes in apparent isolation were also found. Genes occupied 11% of the total sequence, LTR retrotransposons and other repeated elements accounted for 51.9% and the remaining 37.1% could not be annotated.

Chromosomes, Artificial, Bacterial↗

PubMed2002

Physical and genetic mapping of barley (Hordeum vulgare) germin-like cDNAs.

Germin with oxalate oxidase and superoxide dismutase activity is a homohexamer of six manganese-containing interlocked beta-jellyroll monomers with extreme resistance to heat and proteolytic degradation [Woo, E.-J., Dunwell, J. M., Goodenough, P. W., Marvier, A. C. & Pickersill, R. W. (2000) Nat. Struct. Biol. 7, 1036-1038]. This structure is conserved in germin-like proteins (GLPs) with other enzymatic functions and characteristic for proteins deposited in plant cell walls in response to pathogen attack and abiotic stress. Comparative nucleotide and amino acid sequence analyses of 49,610 barley expressed sequence tags identified 124 germin and germin-like cDNAs, which distributed into five subfamilies designated HvGER-I to HvGER-V. Representative cDNAs for these subfamilies hybridized to 67 bacterial artificial chromosome (BAC) clones from a library containing 6.3 genomic equivalents. Twenty-six BAC clones hybridized to the subfamily IV probe and identified a gene-rich region including clone 418E1 of 96 kb encoding eight GLPs (i.e., 1 gene per 12 kb). This BAC clone lacked highly repeated sequences and mapped to the subtelomeric region of the long arm of chromosome 4(4H). Among the six genes of the contig expressed in leaves, one specifies a protein known to be associated with papilla formation in the epidermis upon powdery mildew infection. Three structural genes for oxalate oxidase are present in subfamily I and eight GLPs of various functions in the other subfamilies. These genes map at loci in chromosomes 1(7H), 2 (2H), 3(3H), 4(4H), and 7(5H). Some are present on a single BAC clone. The results are discussed in relation to cereal genome organization.

Chromosome Mapping↗

PubMed2002

LINEs and gypsy-like retrotransposons in Hordeum species.

LINE and gypsy-like retroelements were studied in the genome of Hordeum vulgare, and compared with the representatives of the major sections of the genus Hordeum. We isolated reverse transcriptase (RT) genes from four gypsy-like and three LINE families using PCR primers specific for the corresponding conserved domains. A full-length barley LINE of 6295 bp, named BLIN, was isolated from a BAC genomic library. BLIN looks alien in the barley genome because its G+C content is 62% compared to an average of 45%. The BLIN nucleotide sequence showed it was structurally intact with the features typical of non-LTR retrotransposons, including 16 bp target site duplications, two short cysteine motifs, and two degenerate open reading frames (ORFs). The high degeneracy was also found in RT domain of both gypsy-like and, particularly, LINE families. The copy numbers of the gypsy-like families were relatively low compared to well-characterized copia-like element BARE-1. Each gypsy-like family gave unique RFLP patterns when hybridized to genomic DNA from each of the four basic Hordeum genomes. H. vulgare (I genome) had accumulated more copies than the wild Hordeum species (H, X, Y genomes), with the other I genome species, H. bulbosum, being intermediate. Analysis of the BAC library and in situ hybridization with LINE RT domains showed the low copy number of the LINE families, but there was little correlation between hybridization patterns and the division of the genus into four basic genomes. The distribution and content of gypsy retrotransposons in the BAC library indicated that a few copies are nested, although most are present as single, distinct, copies. Our results suggest that the major groups of retroelements make individual contributions to the shape of the plant genome; the factors involved in their amplification and distribution are independent, also varying among species.

Amino Acid Sequence↗

PubMed2002

Fine mapping of a semi-dwarf gene brachytic 1 in barley.

RFLP markers isolated from barley, wheat and rice were applied to construct a fine structure map of brachytic1, a semi-dwarf gene located on chromosome 1(7H) short arm in barley. The map covered 15.2 cM with the average distance 0.8 cM between markers. A barley cDNA clone, MWG2074B co-segregated with brh1 gene in the test population. Another major band of this clone MWG2074A was 0.8 cM away from brh1 toward centromere. CDO545 and BCD129 were two flanking markers mapped on both sides of brh1, toward distal and pistal, respectively. CDO545 fitted the systenic region of rice genome, chromosome 6 short arm perfectly. However, two major bands of MWG2074 could not be mapped to the target position of rice genome.

Chromosome Mapping↗